詳細

辻内　俊文 （ツジウチ　トシフミ）

  • 理工学部 生命科学科　教授
Last Updated :2024/04/19

コミュニケーション情報 byコメンテータガイド

  • コメント

    肺がんや膵がんを代表とする難治性がんの発生・増殖・浸潤・転移に関わる細胞内分子の同定と、それに基づく新しい分子標的療法開発にむけた基礎的研究を行っています。

研究者情報

学位

  • 医学博士

ホームページURL

J-Global ID

研究キーワード

  • oncology   cancer biology   cell signaling   分子病理学   molecular pathology   

現在の研究分野（キーワード）

    肺がんや膵がんを代表とする難治性がんの発生・増殖・浸潤・転移に関わる細胞内分子の同定と、それに基づく新しい分子標的療法開発にむけた基礎的研究を行っています。

研究分野

  • ライフサイエンス / 実験病理学
  • ライフサイエンス / 人体病理学

学歴

  •         - 1992年   奈良県立医科大学   医学研究科   腫瘍病理学
  •         - 1992年   奈良県立医科大学   Graduate School, Division of Medicine

所属学協会

  • 日本病理学会   日本癌学会   

研究活動情報

論文

  • Regulation of cellular responses to X-ray irradiation through the activation of lysophosphatidic acid (LPA) receptor-3 (LPA3) and LPA2 in osteosarcoma cells
    Ikeda H, Takai M, Yashiro N, Amano Y, Hara K, Yamamoto M, Tsujiuchi T.
    Pathol Res Pract 257 155293  2024年 [査読有り]
  • Roles of lysophosphatidic acid (LPA) receptor-mediated signaling in cellular functions modulated by endothelial cells in pancreatic cancer cells under hypoxic conditions.
    Takai M, Yashiro N, Hara K, Amano Y, Yamamoto M, Tsujiuchi T.
    Pathol Res Pract 255 155192  2024年 [査読有り]
  • Effects of free fatty acid receptor (FFAR) signaling on the modulation of cancer cell functions under hypoxic conditions.
    Yashiro N, Takai M, Yamamoto M; Amano Y; Hara K; Tsujiuchi T.
    Biochem Biophys Res Commun. 699 149554  2024年 [査読有り]
  • Lysophosphatidic acid (LPA) receptor-mediated signaling and cellular responses to anticancer drugs and radiation of cancer cells .
    Ikeda H; Takai M; Tsujiuchi T.
    Adv Biol Regul. 92 101029  2024年 [査読有り]
  • Revisiting ' hallmarks of cancer' in sarcoma.
    Honoki K; Tsujiuchi T; Kishi S; Kuniyasu H
    Journal of Cancer 15 1786 - 1804 2024年 [査読有り]
  • Induction of lysophosphatidic acid (LPA) receptor-mediated signaling regulates cell motility and survival to anticancer drugs in cancer cells treated with hydrogen peroxide.
    Takai M, Takamoto M, Amano Y, Yamamoto M, Hara K, Yashiro N, Tsujiuchi T.
    Adv Biol Regul. 89 100978  2023年 [査読有り]
  • Roles of lysophosphatidic acid (LPA) receptor-2 (LPA2) in the regulation of cellular responses induced by X-ray irradiation and hydrogen peroxide in pancreatic cancer cells
    Okuda A; Takai M; Kurisu R; Takamoto M; Tsujiuchi T
    Int J Radiat Biol 99 1925 - 1933 2023年 [査読有り]
  • Effects of LPA receptor-mediated signaling on the modulation of cellular functions of pancreatic cancer cells cultured in fibroblast supernatants under hypoxic conditions
    Takai M; Okuda A; Amano Y; Yashiro N; Hara K; Yamamoto M; Tsujiuchi T.
    J Bioenerg Biomembr 55 169 - 177 2023年 [査読有り]
  • Takai M; Takauchi M; Kuribayashi M; Tsujiuchi T
    Biochem Biophys Res Commun 661 21 - 27 2023年 [査読有り]
  • Kado T; Kusakari N; Tamaki T; Murota K; Tsujiuchi T; Fukushima N
    Biochem Biophys Res Commun 657 21 24 - 34 2023年 [査読有り]
  • Effects of free fatty acid receptor-2 (FFAR2)-mediated signaling on the regulation of cellular functions in osteosarcoma cells
    Kurisu R; Takai M; Takamoto, M; Tsujiuchi T
    Biochem Biophys Res Commun 646 56 - 62 2023年 [査読有り]
  • Effects of lysophosphatidic acid (LPA) signaling via LPA receptors on cellular functions associated with ATP reduction in osteosarcoma cells treated with ethidium bromide
    Kurisu R; Takamoto M; Minami K; Ueda N; Yamada M; Shima N; Otani T; Sakai Y; Kondo D; Tsujiuchi T
    J Bioenerg Biomembr 54 109 - 117 2022年 [査読有り]
  • Roles of endothelial cells in the regulation of cell motility via lysophosphatidic acid receptor-2 (LPA2) and LPA3 in osteosarcoma cells.
    Minami K; Ueda N; Ishimoto K; Kurisu R; Takamoto M; Ikeda H; Tsujiuchi T
    Exp Mol Pathol 118 104596  2021年 [査読有り]
  • Minami K; Ueda N; Ishimoto K; Kurisu R; Takamoto M; Ikeda H; Tsujiuchi T
    Biochem Biophys Res Commun. 532 3 427 - 432 2020年 [査読有り]
  • Different effects of lysophosphatidic acid receptor-2 (LPA2) and LPA5 on the regulation of chemoresistance in colon cancer cells
    Ishimoto K; Minami A; Minami K; Ueda N; Tsujiuchi T
    J Recept Signal Transduct Res 16 1 - 6 2020年 [査読有り]
  • Lysophosphatidic acid receptor-2 (LPA2)-mediated signaling enhances chemoresistance in melanoma cells treated with anticancer drugs
    Minami K, Ueda N, Ishimoto K, Tsujiuchi T.
    MOLECULAR AND CELLULAR BIOCHEMISTRY 469 89 - 95 2020年 [査読有り]
  • Effects of lysophosphatidic acid (LPA) receptor-2 (LPA2) and LPA3 on the regulation of chemoresistance to anticancer drug in lung cancer cells.
    Ueda N, Minami K, Ishimoto K, Tsujiuchi T.
    Cellular Signalling 69 109551  2020年 [査読有り]
  • Regulation of cell survival through free fatty acid receptor 1 (FFA1) and FFA4 induced by endothelial cells in osteosarcoma cells.
    Minami K, Ueda N, Ishimoto K, Tsujiuchi T.
    J Recept Signal Transduct Res. 40 2 181 - 186 2020年 [査読有り]
  • LPA5-mediated signaling induced by endothelial cells and anticancer drug regulates cellular functions of osteosarcoma cells
    Minami K; Ueda N; Ishimoto K; Tsujiuchi T
    Exp Cell Res. 388 111813  2020年 [査読有り]
  • Modulation of chemoresistance by lysophosphatidic acid (LPA) signaling through LPA5 in melanoma cells treated with anticancer drugs
    Minami K; Ueda N; Maeda H; Ishimoto K; Otagaki S; Tsujiuchi T
    Biochem Biophys Res Commun. 517 359 - 363 2019年 [査読有り]
  • Rapid establishment of highly migratory cells from cancer cells for investigating cellular functions
    Ishimoto K; Minami K; Otagaki S; Tsujiuchi T
    J Recept Signal Transduct Res 39 194 - 198 2019年 [査読有り]
  • Fukushima K; Takahashi K; Kurokawa A; Ishimoto K; Otagaki S; Minami K; Fukushima N; Honoki K; Tsujiuchi T
    Biochem Biophys Res Commun. 496 1 225 - 230 2018年01月 [査読有り]
     
    Lysophosphatidic acid (LPA) signaling through six subtypes of LPA receptors (LPA1 to LPA6) regulates a variety of biological responses in cancer cells. The aim of our study was to evaluate an involvement of LPA receptors in the activation of cell motility by phorbol ester and anticancer drug treatments in melanoma A375 cells. Cells were treated with 12-O-tetradecanoylphorbol- 13-acetate (TPA) and phorbol-12,13-dibutyrate (PDBu) for 3 days. The cell motile activity of TPA treated cells was significantly higher than that of PDBu treated cells, correlating with LPAR5 expression levels. LPA5 knockdown suppressed the high cell motile activity induced by TPA. To assess whether the cell motile activity of A375 cells is stimulated through LPA5 induced by anticancer drugs, the long-term cisplatin (CDDP) and dacarbazine (DTIC) treated cells were generated from A375 cells (A375-CDDP and A375-DTIC cells, respectively). The expression levels of LPA receptor genes were changed in A375-CDDP and A375-DTIC cells. In particular, CDDP and DTIC treatment markedly elevated LPAR5 expressions. The cell motile activities of A375-CDDP and A375-DTIC cells were significantly higher than that of untreated cells. These results suggest that the cell motile activity is regulated through the induction of LPA5 by phorbol ester and anticancer drug treatments in A375 cells.
  • Takahashi K; Fukushima K; Otagaki S; Ishimoto K; Minami K; Fukushima N; Honoki K; Tsujiuchi T
    Journal of Receptors and Signal Transduction 38 1 71 - 75 2018年01月 [査読有り]
     
    Lysophosphatidic acid (LPA) is a simple physiological lipid and exhibits a variety of cellular responses via the activation of G protein-coupled transmembrane LPA receptors (LPA receptor-1 (LPA1) to LPA6). The aim of our study was to investigate effects of LPA receptors on soft agar colony formation in colon cancer cells treated with anticancer drugs. DLD1 cells were treated with fluorouracil (5-FU) or cisplatin (CDDP) for at least six months (DLD-5FU and DLD-CDDP cells, respectively). LPAR1 gene expression was markedly elevated in DLD-5FU cells. In contrast, DLD-CDDP cells showed the high expression of LPAR6 gene. In colony formation assay, DLD-5FU cells formed markedly large-sized colonies, while no colony formation was observed in DLD1 and DLD-CDDP cells. The large-sized colonies formed in DLD-5FU cells were suppressed by LPA1 knockdown. In contrast, LPA6 knockdown increased the size of colonies. In addition, DLD-5FU cells were further treated with CDDP for three months (DLD-C-F cells). DLD-CDDP cells were also treated with 5-FU (DLD-F-C cells). DLD-C-F cells formed large-sized colonies, but not DLD-F-C cells, correlating with LPAR1 and LPAR6 gene expression levels. These results suggest that LPA1 and LPA6 may regulate the colony formation activity in DLD1 cells treated with anticancer drugs.
  • Promotion of cell invasive activity through the induction of LPA receptor-1 in pancreatic cancer cells.
    Fukushima K; Otagaki S; Takahashi K; Minami K; Ishimoto K; Fukushima N; Honoki K; Tsujiuchi T
    J Recept Signal Transduct Res. 38 4 367 - 371 2018年 [査読有り]
  • Lysophosphatidic acid receptor-2 (LPA2) and LPA5 regulate cellular functions during tumor progression in fibrosarcoma HT1080 cells.
    Takahashi K; Minami K; Otagaki S; Ishimoto K; Fukushima K; Fukushima N; Honoki K; Tsujiuchi T
    Biochem Biophys Res Commun. 503 2698 - 2703 2018年 [査読有り]
  • Takahashi K, Fukushima K, Tanaka K, Minami K, Ishimoto K, Otagaki S, Fukushima N, Honoki K, Tsujiuchi T.
    Experimental Cell Research 369 2 316 - 324 2018年 [査読有り]
     
    Lysophosphatidic acid (LPA) signaling via G protein-coupled LPA receptors mediates various biological effects in cancer cells. This study aimed to investigate the roles of LPA receptors in the regulation of cellular functions during tumor progression in osteosarcoma cells. Long-term cisplatin (CDDP)-treated MG63-C and MG63-R7-C cells were generated from osteosarcoma MG-63 and highly-migratory MG63-R7 cells, respectively. LPAR2 and LPAR3 expression levels were significantly higher in MG63-C cells than in MG-63 cells, while LPAR1 expression was reduced. MG63-C cells were highly motile, compared with MG-63 cells. MG63-C cell motility was suppressed by LPA2 knockdown and enhanced by the LPA1/LPA3 antagonist, dioctanoylglycerol pyrophosphate. LPAR2 and LPAR3 expression levels were significantly elevated in MG63-R7-C cells in comparison with MG63-R7 cells. MG63-R7-C cells were found to be highly invasive, correlating with metalloproteinase-2 activation. MG63-R7-C cells formed large colonies, whereas colony formation was absent from MG63-R7 cells. Notably, MG63-R7-C cell activities were inhibited by LPA2 knockdown. These results suggest that LPA signaling via LPA2 plays an important role in the acquisition of malignant properties during tumor progression in MG-63 cells.
  • Induction of GPR40 positively regulates cell motile and growth activities in breast cancer MCF-7 cells
    Fukushima K; Takahashi K; Kusaka M; Ishimoto K; Otagaki S; Minami K; Fukushima N; Honoki K; Tsujiuchi T
    J Recept Signal Transduct Res 38 4 311 - 315 2018年 [査読有り]
  • Involvement of FFA1 and FFA4 in the regulation of cellular functions during tumor progression in colon cancer cells.
    Takahashi K; Fukushima K; Onishi Y; Minami K; Otagaki S; Ishimoto K; Fukushima N; Honoki K; Tsujiuchi T
    Exp Cell Res 369 54 - 60 2018年 [査読有り]
  • Yuhei Irie; Maho Tsubota; Hiroyasu Ishikura; Fumiko Sekiguchi; Yuka Terada; Toshifumi Tsujiuchi; Keyue Liu; Masahiro Nishibori; Atsufumi Kawabata
    JOURNAL OF NEUROIMMUNE PHARMACOLOGY 12 4 693 - 707 2017年12月 [査読有り]
     
    Extracellular high mobility group box 1 (HMGB1) activates the receptor for advanced glycation end products (RAGE) or Toll-like receptor 4 (TLR4) and forms a heterocomplex with CXCL12 that strongly activates CXCR4, promoting inflammatory and pain signals. In the present study, we investigated the role of HMGB1 in pancreatic pain accompanying cerulein-induced acute pancreatitis in mice. Abdominal referred hyperalgesia accompanying acute pancreatitis occurred within 1 h after 6 hourly injections of cerulein. The anti-HMGB1 neutralizing antibody or recombinant human soluble thrombomodulin (rhsTM), known to inactivate HMGB1, abolished the cerulein-induced referred hyperalgesia, but not pancreatitis itself. Plasma or pancreatic HMGB1 levels did not change, but macrophage infiltration into the pancreas occurred 1 h after cerulein treatment. Minocycline, a macrophage/microglia inhibitor, ethyl pyruvate that inhibits HMGB1 release from macrophages, or liposomal clodronate that depletes macrophages prevented the referred hyperalgesia, but not pancreatitis. Antagonists of RAGE or CXCR4, but not TLR4, strongly suppressed the cerulein-induced referred hyperalgesia, but not pancreatitis. Upregulation of RAGE, CXCR4 and CXCL12, but not TLR4, were detected in the pancreas 1 h after cerulein treatment. Our data suggest that HMGB1 regionally secreted by macrophages mediates pancreatic pain by targeting RAGE and CXCL12/CXCR4 axis in the early stage of acute pancreatitis.
  • Aiko Tanaka; Akane Yamamoto; Kaeko Murota; Toshifumi Tsujiuchi; Masao Iwamori; Nobuyuki Fukushima
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 493 1 468 - 473 2017年11月 [査読有り]
     
    Free fatty acids not only play a role in cell membrane construction and energy production but also exert diverse cellular effects through receptor and non-receptor mechanisms. Moreover, epidemiological and clinical studies have so far suggested that polyunsaturated fatty acids (PUFAs) could have health benefits and the advantage as therapeutic use in cancer treatment. However, the underlying mechanisms of PUFA-induced cellular effects remained to be cleared. Here, we examined the effects of omega-3 and to omega-6 PUFAs on cell death in ovarian cancer cell lines. omega-3 PUFA, docosahexaenoic acid (DHA) and omega-6 PUFA, gamma-linolenic acid (gamma-LNA) induced cell death in KF28 cells at the levels of physiological concentrations, but not HAC2 cells. Pharmacological and biochemical analyses demonstrated that cell death induced by DHA and gamma-LNA was correlated with activation of JNK and p38 MAP kinases, and further an upstream MAP kinase kinase, apoptosis signal -regulating kinase 1, which is stimulated by reactive oxygen species (ROS). Furthermore, an antioxidant vitamin E attenuated PUFA-induced cell death and MAP kinase activation. These findings indicate that PUFA-induced cell death involves ROS-dependent MAP kinase activation and is a cell type -specific action. A further study of the underlying mechanisms for ROS-dependent cell death induced by PUFAs will lead to the discovery of a new target for cancer therapy or diagnosis. (C) 2017 Elsevier Inc. All rights reserved.
  • Kaede Takahashi; Kaori Fukushima; Nobuyuki Fukushima; Kanya Honoki; Toshifumi Tsujiuchi
    MOLECULAR AND CELLULAR BIOCHEMISTRY 431 1-2 29 - 35 2017年07月 [査読有り]
     
    Lysophosphatidic acid (LPA) is a simple biophysical lipid which interacts with at least six subtypes of G protein-coupled LPA receptors (LPA(1)-LPA(6)). In cancer cells, LPA signaling via LPA receptors is involved in the regulation of malignant properties, such as cell growth, motility, and invasion. The aim of this study was to assess whether LPA receptors regulate cellular functions of fibrosarcoma cells treated with anticancer drug. HT1080 cells were maintained by the stepwise treatment of cisplatin (CDDP) at a range of 0.01 to 1.0 A mu M for approximately 6 months. The cell motile and invasive activities of long-term CDDP-treated (HT-CDDP) cells were significantly stimulated by LPA treatment, while HT-CDDP cells in the static state showed the low cell motile and invasive activities in comparison with HT1080 cells. Since the expression level of LPAR2 gene was markedly elevated in HT-CDDP cells, LPA(2) knockdown cells were generated from HT-CDDP cells. The cell motile and invasive activities of HT-CDDP cells were reduced by LPA(2) knockdown. In colony assay, large-sized colonies formed by long-term CDDP treatment were suppressed by LPA(2) knockdown. In addition, LPA(2) knockdown cells reduced LPA production by autotaxin (ATX), correlating with ATX expression level. These results suggest that LPA signaling via LPA(2) may play an important role in the regulation of cellular functions in HT1080 cells treated with CDDP.
  • Shoichi Ishii; Toshifumi Tsujiuchi; Nobuyuki Fukushima
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 486 3 767 - 773 2017年05月 [査読有り]
     
    Lysophosphatidic acid (LPA), an extracellular lipid mediator, exerts various cellular effects through activation of LPA receptors, LPA(1)-LPA(6), in many types of cells including cancer cells. We recently found several missense mutations of Lpar1 in rat cancer tissues. One of these mutations is located at the extracellular tip of the seventh transmembrane domain of LPA(1), and another three mutations are found within the NPXXY motif in the seventh transmembrane domain. These mutants are designated F295S LPA(1) and P308S, 1310T, and Y311H LPA(1), respectively. Here, we examined the functions of these LPA(1) mutants. Compared with wild-type (WT) LPA(1), F295S, P308S, and 1310T LPA(1) showed decreased maximal responses in inhibition of cAMP formation, Ca2+ mobilization, and cytoskeletal changes. Y311H LPA(1) failed to show LPA-induced cellular responses. However, these LPA(1) mutants were internalized in response to LPA exposure. Finally, while WT and F295S LPA(1) showed a similar, broad distribution throughout the cell, P308S, 1310T, and Y311H LPA(1) displayed a restricted cellular distribution and co-localized with the endoplasmic reticulum. These data suggest that the LPA(1) mutants perturb LPA signaling in cancer tissues. (C) 2017 Elsevier Inc. All rights reserved.
  • Kaede Takahashi; Kaori Fukushima; Yuka Onishi; Yusuke Node; Karin Inui; Nobuyuki Fukushima; Kanya Honoki; Toshifumi Tsujiuchi
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 484 3 675 - 680 2017年03月 [査読有り]
     
    G-protein-coupled receptor 120 (GPR120) and GPR40 are members of free fatty acid (FFA) receptors and mediate a variety of biological responses through binding of medium- and long -chain FFAs. Recently, it has been reported that GPR120 and GPR40 regulated cellular functions of cancer cells. In the present study, to assess whether GPR120 and GPR40 are involved in the enhancement of cell motile activity of osteosarcoma cells, we established highly migratory (MG63-R7) cells from osteosarcoma MG -63 cells. The expression level of GPR120 gene was significantly higher in MG63-R7 cells than in MG -63 cells, while no change of GPR40 expression was observed. In cell motility assay, the cell motile activity of MG63-R7 cells was approximately 200 times higher than that of MG-63 cells. The cell motile activity of MG63-R7 cells was stimulated by GW9508, which is an agonist of GPR120 and GPR40. Moreover, a GPR40 antagonist GW1100 elevated the cell motile activity of MG63-R7 cells in the presence of GW9508. To confirm the effects of GPR120 and GPR40 on the cell motile activity of MG63-R7 cells, GPR120 knockdown cells were generated from MG63-R7 cells. The cell motile activity of MG63-R7 cells was markedly suppressed by GPR120 knockdown. These results indicated that GPR120 enhanced and GPR40 inhibited the cell motile activity of highly migratory osteosarcoma cells. (C) 2017 Elsevier Inc. All rights reserved.
  • Kaori Fukushima; Kaede Takahashi; Eri Yamasaki; Yuka Onishi; Nobuyuki Fukushima; Kanya Honoki; Toshifumi Tsujiuchi
    EXPERIMENTAL CELL RESEARCH 352 1 139 - 145 2017年03月 [査読有り]
     
    Lysophosphatidic acid (LPA) signaling via G protein -coupled LPA receptors exhibits a variety of biological effects, such as cell proliferation, motility and differentiation. The aim of this study was to evaluate the roles of LPA(1) and LPA(3) in cellular functions during tumor progression in pancreatic cancer cells. LPA(1) and LPA(3) knockdown cells were generated from PANC-1 cells. The cell motile and invasive activities of PANC-1 cells were inhibited by LPA(1) and LPA(3) knockdown. In gelatin zymography, LPA(1) and LPA(3) knockdown cells indicated the low activation of matrix metalloproteinase-2 (MMP-2) in the presence of LPA. Next, to assess whether LPA1 and LPA3 regulate cellular functions induced by anticancer drug, PANC-1 cells were treated with cisplatin (CDDP) for approximately 6 months. The cell motile and invasive activities of long-term CDDP treated cells were markedly higher than those of PANC-1 cells, correlating with the expression levels of LPARI and LPAR3 genes. In soft agar assay, the long-term CDDP treated cells formed markedly large sized colonies. In addition, the cell motile and invasive activities enhanced by CDDP were significantly suppressed by LPA(1) and LPA(3) knockdown as well as colony formation. These results suggest that LPA signaling via LPA(1) and LPA(3) play an important role in the regulation of cellular functions during tumor progression in PANC-1 cells.
  • Kaede Takahashi; Kaori Fukushima; Yuka Onishi; Karin Inui; Yusuke Node; Nobuyuki Fukushima; Kanya Honoki; Toshifumi Tsujiuchi
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 483 1 652 - 657 2017年01月 [査読有り]
     
    Lysophosphatidic acid (LPA) is an extracellular biological lipid and interacts with six subtypes of G protein-coupled LPA receptors (LPA(1) to LPA(6)). LPA receptors exhibit a variety of cellular functions, depending on types of cancer cells. In this study, to assess the roles of LPA(4) and LPA(6) in cell growth and motile activities of colon cancer cells, LPA(4) and LPA(6) knockdown cells were established from DLD1 and HCT116 cells. LPA treatment increased the cell growth activities of LPA(4) and LPA(6) knockdown cells, compared with control cells. The cell motile activities of LPA(4) and LPA(6) knockdown cells were significantly higher than those of control cells. To evaluate the effects of LPA(4) and LPA(6) on cell motile activity induced by anticancer drug, long-term fluorouracil (5-FU) treated (DLD-5FU) cells were generated. The expression levels of LPAR1, LPAR4 and LPAR6 genes were significantly increased in DLD-5FU cells. DLD5FU cells showed the high cell motile activity, compared with DLD1 cells. The increased cell motile activity was markedly stimulated by LPA(4) and LPA(6) knockdown. In contrast, the cell motile activity enhanced by 5-FU treatment was suppressed by LPA(1) knockdown. These results suggest that LPA signaling via LPA(4) and LPA(6) negatively regulates the cell motile activities of DLD1 and HCT116 cells as well as long-term 5-FU treated cells. (C) 2016 Elsevier Inc. All rights reserved.
  • Shuhei Ishii; Yuka Kitamura; Miku Hirane; Ayaka Tomimatsu; Kaori Fukushima; Kaede Takahashi; Nobuyuki Fukushima; Kanya Honoki; Toshifumi Tsujiuchi
    MOLECULAR CARCINOGENESIS 55 11 1553 - 1559 2016年11月 [査読有り]
     
    G-protein-coupled receptor 40 (GPR40) and GPR120 mediate a variety of biological functions by the binding of long and medium chain free fatty acids. In the present study, we investigated a role of GPR40 in the pathogenesis of fibrosarcoma HT1080 cells. The GPR40 gene expression was detected in HT1080 cells, but not the GPR120 gene. The cell motile and invasive activities were markedly enhanced by GPR40 knockdown, compared with control cells. To evaluate whether GPR40 is involved in the cellular functions of HT1080 cells during anticancer drug treatment, HT1080 cells were maintained in condition medium containing cisplatin (CDDP) (0.01-1.0 mu M) for 6 mo. The expression levels of the GPR40 gene was elevated by the long-term CDDP treatment in HT1080 cells, while the GPR120 gene expression remained unchanged. The cell motile and invasive activities of HT1080 cells treated with CDDP were significantly lower than those of untreated cells. In gelatin zymography, the activities of matrix metalloproteinase-2 (MMP-2) and MMP-9 of HT1080 cells were enhanced by the long-term CDDP treatment. In addition, GW9508 which is an agonist of GPR40 and GPR120 suppressed the cell motile and invasive activities of HT1080 cells treated with CDDP as well as the MMP activation. These results suggest that GPR40 negatively regulates the tumor progression of fibrosarcoma cells. (C) 2015 Wiley Periodicals, Inc.
  • Miku Hirane; Shuhei Ishii; Ayaka Tomimatsu; Kaori Fukushima; Kaede Takahashi; Nobuyuki Fukushima; Kanya Honoki; Toshifumi Tsujiuchi
    MOLECULAR CARCINOGENESIS 55 11 1573 - 1583 2016年11月 [査読有り]
     
    Lysophosphatidic acid (LPA) signaling via LPA receptors (LPA1 to LPA6) mediates a variety of cellular functions, including cell motility. In the present study, we investigated the effects of LPA receptors on cell motile activity during multi-stage hepatocarcinogenesis in rat liver epithelial WB-F344 cells treated with chemical liver carcinogens. Cells were treated with a initiator (N-nitrosodiethylamine (DEN)) and three promoters (phenobarbital (PB), okadaic acid (OA) and clofibrate) every 24 h for 2 days. Cell motile activity was elevated by DEN, correlating with Lpar3 expression. PB, OA, and clofibrate elevated Lpar1 expression and inhibited cell motile activity. To evaluate the effects of long-term treatment on cell motility, cells were treated with DEN and/or PB for at least 6 months. Lpar3 expression and cell motile activity were significantly elevated by the long-term DEN treatment with or without further PB treatment. In contrast, long-term PB treatment with or without further DEN elevated Lpar1 expression and inhibited cell motility. When the synthesis of extracellular LPA was blocked by a potent ATX inhibitor S32826 before cell motility assay, the cell motility induced by DEN and PB was markedly suppressed. These results suggest that activation of the different LPA receptors may regulate the biological functions of cells treated with chemical carcinogens. (C) 2015 Wiley Periodicals, Inc.
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    岸 真五; 朴木 寛弥; 藤井 宏真; 塚本 真治; 城戸 顕; 藤原 里奈; 森 汐莉; 門地 優衣; 羅 奕; 笹平 智則; 辻内 俊文; 田中 康仁; 国安 弘基
    日本癌学会総会記事 75回 J - 3094 2016年10月
  • 岸 真五; 國安 弘基; 朴木 寛弥; 塚本 真治; 城戸 顕; 藤井 宏真; 近藤 裕美子; 羅 奕; 門地 優衣; 森 汐莉; 笹平 智則; 辻内 俊文; 田中 康仁
    日本整形外科学会雑誌 90 6 S1215 - S1215 (公社)日本整形外科学会 2016年06月
  • Kaori Fukushima; Kaede Takahashi; Nobuyuki Fukushima; Kanya Honoki; Toshifumi Tsujiuchi
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 475 1 25 - 30 2016年06月 [査読有り]
     
    G-protein-coupled receptor 120 (GPR120) and GPR40 exhibit a variety of biological responses by the binding of free fatty acids. 12-O-Tetradecanoylphorbol-13-acetate (TPA) is a tumor promoting agent of skin carcinogenesis. It is known that TPA treatment stimulates cell motile activity of cancer cells, including melanoma cells. In the present study, we investigated whether GRP120 and GPR40 are involved in regulation of cell motile activity induced by TPA in two melanoma cell lines. A375 and G361 cells were treated with TPA at a concentration of 10 nM for 24 h. The cell motile activity of A375 cells was significantly increased by TPA, correlating with GPR40 expression. In contrast, TPA suppressed the cell motile activity of G361 cells, while GPR120 and GPR40 expressions were increased. The cell motile activity of A375 cells treated with TPA was markedly increased by GPR120 knockdown. In addition, to assess roles of GPR120 and GPR40 in cellular functions of A375 cells by the long-term TPA treatment, cells were treated with TPA (1 nM) for at least 3 months. The long-term TPA treatment induced the high cell motile activity and elevated GPR120 and GPR40 expressions. The high cell motile activity of A375 cells stimulated by the long-term TPA treatment was enhanced by GPR120 knockdown. These results suggest that GPR120 negatively and GPR40 positively regulate cell motile activities induce by TPA in melanoma cells. (C) 2016 Elsevier Inc. All rights reserved.
  • Tsubasa Kita; Yui Kadochi; Kaede Takahashi; Kaori Fukushima; Eri Yamasaki; Taiki Uemoto; Miku Hirane; Nobuyuki Fukushima; Kanya Honoki; Toshifumi Tsujiuchi
    EXPERIMENTAL CELL RESEARCH 342 2 193 - 199 2016年03月 [査読有り]
     
    Free fatty acids (FFAs) are dietary nutrients which mediate a variety of biological effects through binding to G-protein-coupled FFA receptors (FFARs). G-protein-coupled receptor 120 (GPR120) and GPR40 are identified as FFARs for long- and medium-chain fatty acids. Here we investigated whether GPR120 and GPR40 are involved in the acquisition of malignant properties in lung cancer cells. Three lung cancer RLCNR, LL/2 and A549 cells used in this study expressed GPR120 and GPR40 genes. The cell motile activities of all cells were significantly suppressed by a GPR40 antagonist GW1100. In addition, GPR40 knockdown inhibited the cell motile activity of A549 cells. In gelatin zymography, matrix metalloproteinase-2 (MMP-2) activity in GPR40 knockdown was significantly lower than that in control cells. Next, to evaluate effects of GPR120 and GPR40 on cellular functions induced by anti-cancer drug, the long-term cisplatin (CDDP) treated (A549-CDDP) cells were generated. The expression levels of GPR120 and GPR40 were significantly decreased in A549-CDDP cells. While A549-CDDP cells showed the high cell motile activity, GW1100 suppressed the cell motile activity of A549-CDDP cells. These results demonstrate that GPR120 negatively and GPR40 positively regulate cellular functions during tumor progression in lung cancer cells. (C) 2016 Elsevier Inc. All rights reserved.
  • Crossroads of hallmarks in aging and cancer: Anti-aging and anti-cancer target pathways can be shared?
    Honoki K; Kuniyasu H; Kishi S; Tanaka Y; Tsujiuchi T
    Trends Cancer Res 11 41 - 59 2016年 [査読有り]
  • Shiori Mori; Mutsumi Araki; Shuhei Ishii; Miku Hirane; Kaori Fukushima; Ayaka Tomimatsu; Kaede Takahashi; Nobuyuki Fukushima; Toshifumi Tsujiuchi
    MOLECULAR AND CELLULAR BIOCHEMISTRY 408 1-2 147 - 154 2015年10月 [査読有り]
     
    Lysophosphatidic acid (LPA) signaling via LPA receptors provides a variety of cellular functions, including angiogenesis. In this study, to assess an involvement of LPA receptors in cell motile activities of endothelial cells during chemotherapy, F-2 cells were treated with cisplatin (CDDP) and doxorubicin (DOX) at a concentration of 0.01 mu M every 24 h for at least 1 month. The treatment of CDDP and DOX inhibited the expression levels of the LPA receptor-1 (Lpar1), Lpar2, and Lpar3 genes in F-2 cells. The cell motile activities of CDDP and DOX treated cells were relatively lower than those of untreated cells. Next, we investigated whether cancer cells could stimulate the cell motile activities of F-2 cells treated with CDDP and DOX. For cell motility assay, CDDP- and DOX-treated cells were co-cultured with pancreatic cancer PANC-1 cells. The cell motile activities of CDDP- and DOX-treated cells were significantly enhanced by the existence of PANC-1 cells, correlating with the LPA receptor expressions. In addition, the elevated cell motile activities were suppressed by the pretreatment of an autotaxin inhibitor S32826. These results suggest that LPA signaling via LPA receptors may regulate the cell motile activities of F-2 cells treated with anticancer drugs.
  • Kaori Fukushima; Eri Yamasaki; Shuhei Ishii; Ayaka Tomimatsu; Kaede Takahashi; Miku Hirane; Nobuyuki Fukushima; Kanya Honoki; Toshifumi Tsujiuchi
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 465 3 512 - 515 2015年09月 [査読有り]
     
    Free fatty acids (FFAs) act as extracellular signaling molecules through binding to G-protein-coupled FFA receptors (FFARs). GPR120 and GPR40 are identified as FFARs for medium- and long-chain fatty acids. In the present study, we investigated roles of GPR120 and GPR40 in cellular functions of pancreatic cancer PANC-1 cells, using GPR120 and GPR40 knockdown cells (PANC-sh120 and PANC-sh40 cells respectively). In cell motility assay, PANC-sh120 cells showed the low cell motility, compared with control cells. In contrast, the cell motility of PANC-sh40 cells was significantly higher than that of control cells. Activity levels of matrix metalloproteinases (MMPs) were measured by gelatin zymography. While PANC-sh120 cells indicated the reduced MMP-2 activity, MMP-2 activity in PANC-sh40 cells was significantly higher than that in control cells. On the other hand, no activation of MMP-9 was detected in all cells. In colony assay, the large sized colonies were markedly formed in PANC-sh40 cells. No colony formation was observed in PANC-sh120 cells as well as control cells. These results suggest that distinct effects of GPR120 and GPR40 are involved in the acquisition of malignant property in pancreatic cancer cells. (C) 2015 Elsevier Inc. All rights reserved.
  • Nobuyuki Fukushima; Shoichi Ishii; Toshifumi Tsujiuchi; Nao Kagawa; Kazutaka Katoh
    CELLULAR AND MOLECULAR LIFE SCIENCES 72 12 2377 - 2394 2015年06月 [査読有り]
     
    Lysophosphatidic acid (LPA) is a bioactive lipid mediator that activates G protein-coupled LPA receptors to exert fundamental cellular functions. Six LPA receptor genes have been identified in vertebrates and are classified into two subfamilies, the endothelial differentiation genes (edg) and the non-edg family. Studies using genetically engineered mice, frogs, and zebrafish have demonstrated that LPA receptor-mediated signaling has biological, developmental, and pathophysiological functions. Computational analyses have also identified several amino acids (aa) critical for LPA recognition by human LPA receptors. This review focuses on the evolutionary aspects of LPA receptor-mediated signaling by comparing the aa sequences of vertebrate LPA receptors and LPA-producing enzymes; it also summarizes the LPA receptor-dependent effects commonly observed in mouse, frog, and fish.
  • Shuhei Ishii; Miku Hirane; Kaori Fukushima; Ayaka Tomimatsu; Nobuyuki Fukushima; Toshifumi Tsujiuchi
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 461 1 59 - 64 2015年05月 [査読有り]
     
    Lysophosphatidic acid (LPA) is an extracellular biological lipid which interacts with G protein-coupled LPA receptors (LPA(1) to LPA(6)). LPA signaling via LPA receptors mediates several cellular responses. In the present study, to assess the roles of LPA(4), LPA(6) and LPA(6) in cellular functions of pancreatic cancer cells, we generated LEA receptor knockdown cells from PANC-1 cells (PANC-sh4, PANC-sh5 and PANC-sh6 cells, respectively). In cell motility assay, PANC-sh4 and PANC-sh5 cells enhanced the cell motile activities, compared with control cells. In contrast, the cell motile activity of PANC-sh6 cells was suppressed. The invasive activities of PANC-sh4 and PANC-sh5 cells were markedly stimulated, while PANC-sh6 cells showed the low invasive activity. In colony assay, PANC-sh4 and PANC-sh5 cells formed the large sized colonies, but not PANC-sh6 cells. When endothelial cells were incubated with supernatants from PANC-sh4 and PANC-sh5 cells, the cell motility and tube formation of endothelial cells were significantly induced, but not PANC-sh6 cells. These results suggest that the diverse roles of LPA(4), LPA(6) and LPA(6) are involved in the activation of tumor progression in pancreatic cancer cells. (C) 2015 Elsevier Inc. All rights reserved.
  • Shuhei Ishii; Miku Hirane; Yuka Kitamura; Shiori Mori; Nobuyuki Fukushima; Kanya Honoki; Toshifumi Tsujiuchi
    MOLECULAR AND CELLULAR BIOCHEMISTRY 400 1-2 145 - 151 2015年02月 [査読有り]
     
    G-protein-coupled receptor 120 (GPR120) is identified as a G-protein-coupled receptor for unsaturated long-chain free fatty acids that mediates insulin signaling and anti-inflammatory effects. Recently, it has been reported that GPR120 promotes the cell motile activity and angiogenesis in cancer cells. In this study, we assessed the role of GPR120 in the cell motile activity induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in rat liver epithelial WB-F344 cells. Cells were treated with TPA at a concentration of 5 nM for 72 h. The expression level of the Gpr120 gene was measured by quantitative real-time RT-PCR analysis. Cells treated with TPA showed the elevated Gpr120 expression, in comparison with untreated cells. In cell motility assays, the cell motile activity of cells treated with TPA was significantly higher than that of untreated cells. To confirm whether GPR120 is involved in the cell motile activity mediated by TPA, we generated GPR120 knockdown cells from WB-F344 cells. The cell motile activity induced by TPA was significantly suppressed by GPR120 knockdown. These results suggest that GPR120 plays an important role in the cell motile activity induced by TPA in WB-F344 cells.
  • Shuhei Ishii; Miku Hirane; Sayumi Kato; Nobuyuki Fukushima; Toshifumi Tsujiuchi
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 456 1 135 - 138 2015年01月 [査読有り]
     
    Free fatty acids (FFAs) are dietary nutrients which act as ligands for FFAs receptors. G-protein-coupled receptor 120 (GPR120) and GPR40 are activated by long and medium chain FFAs. In the present study, we investigated the role of the GPR120 and GPR40 in cell motile activity stimulated by ethionine in rat liver epithelial WB-F344 cells. Cells were treated with ethionine at a concentration of 10 mu M every 24 h for 2 days. The expression levels of the Gpr120 and Gpr40 genes in WB-F344 cells treated ethionine were significantly higher than those in untreated cells. In cell motility assay, the cell motile activity of WB-F344 cells was markedly elevated by ethionine, compared with untreated cells. To evaluate the effects of GPR120 on the cell motile activity by ethionine, we established GPR120 knockdown cells from WB-F344 cells. The cell motile activity stimulated by ethionine was significantly suppressed by GPR120 knockdown. In addition, a potent GPR40 antagonist GW1100 enhanced the cell motile activity by ethionine. These results suggest that opposite effects of GPR120 and GPR40 may be involved in the cell motile activity stimulated by ethionine in WB-F344 cells. (C) 2014 Elsevier Inc. All rights reserved.
  • Yuji Morimoto; Shoichi Ishii; Jun-ichi Ishibashi; Kazutaka Katoh; Toshifumi Tsujiuchi; Nao Kagawa; Nobuyuki Fukushima
    GENE 551 2 189 - 200 2014年11月 [査読有り]
     
    Lysophosphatidic acid (LPA) signaling is known to play biological and pathophysiological roles in many types of animals. Medaka (Oryzias latipes) is an experimental fish that can be easily maintained, propagated, and analyzed, and whose genome has been completely sequenced. However, there is limited information available regarding medaka LPA receptors. Here, using information from the medaka genome database, we examine the genomic structures, expression, and functions of six LPA receptor genes, Lpar1-Lpar6. Our analyses reveal that the genomic structures of Lpar1 and Lpar4 are different from those deduced from the database. Functional analyses using a heterologous expression system demonstrate that all medaka LPA receptors except for LPA(5b) respond to LPA treatment with cytoskeletal changes. These findings provide useful information on the structure and function of medaka LPA receptor genes, and identify medaka as a useful experimental model for exploration of the biological significance of LPA signaling. (C) 2014 Elsevier B.V. All rights reserved.
  • Yan Dong; Miku Hirane; Mutsumi Araki; Nobuyuki Fukushima; Kanya Honoki; Toshifumi Tsujiuchi
    MOLECULAR AND CELLULAR BIOCHEMISTRY 393 1-2 17 - 22 2014年08月 [査読有り]
     
    LPA signaling via LPA receptors [LPA receptor-1 (LPA(1))-LPA(6)] mediates the several cellular responses in cancer cells, including cell motility and invasion. In the present study, to investigate a role of LPA(5) in the cell motile and invasive activities of sarcoma cells, LPAR5 knockdown (HOSL5 and HT1080L5) cells were generated from human osteosarcoma HOS and fibrosarcoma HT1080 cells, respectively. In cell motility assays with cell culture inserts, HOSL5 and HT1080L5 cells indicated the high cell motile activities, compared with control cells. The cell invasive activities of HOSL5 and HT1080L5 cells were significantly higher than those of control cells. Moreover, the activities of matrix metalloproteinase (MMP)-2 and MMP-9 were measured by gelatin zymography. MMP-2 was significantly activated in HOSL5 cells, but not MMP-9. The elevated activities of MMP-2 and MMP-9 were found in HT1080L5 cells, in comparison with control cells. These results suggest that LPA signaling via LPA(5) negatively regulates the cell motile and invasive activities of human sarcoma cells.
  • Yan Dong; Mutsumi Araki; Miku Hirane; Eriko Tanabe; Nobuyuki Fukushima; Toshifumi Tsujiuchi
    JOURNAL OF RECEPTORS AND SIGNAL TRANSDUCTION 34 3 201 - 204 2014年06月 [査読有り]
     
    Lysophosphatidic acid (LPA) signaling via G protein-coupled transmembrane LPA receptors (LPA(1) to LPA(6)) mediates a variety of cellular functions, including cell proliferation, migration, morphogenesis, and differentiation. Recently, we demonstrated that the different induction of LPA receptors by estrogens regulates cell motile activity of rat liver epithelial WB-F344 cells. In the present study, to assess whether endocrine disruptors (EDs) are involved in cellular functions through LPA signaling, we measured cell motile activity and LPA receptor expressions in WB-F344 cells treated with bisphenol A (BPA) and 4-nonylphenol (4-NP). Using quantitative real time RT-PCR analysis, the Lpar1 expression was elevated in BPA-treated cells, whereas the Lpar3 expression was decreased. In contrast, 4-NP increased the Lpar3 expression, but not the Lpar1 and Lpar2. For cell motility assay with a Cell Culture Insert, cell motile activity of BPA-treated cells was significantly lower than that of untreated cells. In contrast, 4-NP markedly enhanced cell motile activity. The effects of BPA and 4-NP on cell motility were inhibited by the Lpar1 or Lpar3 knockdown. These results suggest that BPA and 4-NP may regulate cell motile activity through the different induction of LPA receptors in WB-F344 cells.
  • Mutsumi Araki; Misaho Kitayoshi; Yan Dong; Miku Hirane; Shuhei Ozaki; Shiori Mori; Nobuyuki Fukushima; Kanya Honoki; Toshifumi Tsujiuchi
    GROWTH FACTORS 32 3-4 117 - 122 2014年06月 [査読有り]
     
    Lysophosphatidic acid (LPA) is a bioactive lipid that interacts with G protein-coupled LPA receptors (LPA receptor-1 (LPA(1)) to LPA(6)). Here, we investigated the effects of LPA signaling via LPA(5) on cellular functions of sarcoma cells by generating Lpar5 overexpressing and Lpar5 knockdown cells from rat osteosarcoma and malignant fibrous histiocytoma cells, respectively. The cell motility activity of Lpar5 overexpressing cells was significantly lower, while Lpar5 knockdown cells showed high cell motility, compared with respective controls. Gelatin zymography showed that LPA(5) suppressed the activation of matrix metalloproteinase-2. LPA(5) also inhibited the cell motility activity of endothelial cells, correlating with the expression levels of vascular endothelial growth factor genes. These results suggest that LPA signaling via LPA(5) negatively regulates the cellular functions of rat sarcoma cells.
  • Toshifumi Tsujiuchi; Miku Hirane; Yan Dong; Nobuyuki Fukushima
    JOURNAL OF RECEPTORS AND SIGNAL TRANSDUCTION 34 3 149 - 153 2014年06月 [査読有り]
     
    Lysophosphatidic acid (LPA) is a bioactive lipid that interacts with G protein-coupled transmembrane LPA receptors (LPA(1) to LPA(6)). LPA mediates a variety of cellular responses in normal cells, including cell growth, motility, differentiation, morphogenesis, and prevention from apoptosis. Furthermore, LPA signaling via LPA receptors is involved in the pathogenesis of several diseases, including cancer. Cell motility is one of the important properties during the progression of cancer cells. In recent studies, it has been demonstrated that LPA receptors have the diverse effects in the cell motile activities of cancer cells, depending on the cell types involved. In this review, we provide the current knowledge for the biological roles of LPA receptors in the cell motile activities of cancer cells.
  • Junichi Tanaka; Kaoru Yamaguchi; Hiroyasu Ishikura; Maho Tsubota; Fumiko Sekiguchi; Yukari Seki; Toshifumi Tsujiuchi; Akira Murai; Takehiro Umemura; Atsufumi Kawabata
    NEUROPHARMACOLOGY 79 112 - 118 2014年04月 [査読有り]
     
    High mobility group box 1 (HMGB1), one of damage-associated molecular patterns (DAMPs), plays roles in not only inflammation but also processing of somatic pain. Given that no evidence for roles of HMGB1 in visceral pain signaling is available, we asked if HMGB1 participates in bladder pain accompanying cystitis caused by cyclophosphamide in mice, using the anti-HMGB1 neutralizing antibody and recombinant human soluble thrombomodulin (rhsTM) that sequesters HMGB1 and promotes its degradation by thrombin. Cyclophosphamide, administered i.p., caused bladder pain-like nociceptive behavior and referred hyperalgesia accompanying cystitis symptoms including increased bladder weight, an indicator of edema, in mice. The cyclophosphamide-induced bladder pain and referred hyperalgesia, but not increased bladder weight, were prevented by i.p. preadministration of the anti-HMGB1 neutralizing antibody or rhsTM. HMGB1, given i.p., facilitated the bladder pain and referred hyperalgesia caused by a subeffective dose of cyclophosphamide, an effect blocked by rhsTM. In the cyclophosphamide-treated mice, HMGB1 levels greatly decreased in the bladder tissue, particularly in the urothelial cells, but did not change in the plasma. Low molecular weight heparin, known to inhibit the receptor for advanced glycation end products (RAGE), but not lipopolysaccharide from Rhodobacter sphaeroides, an inhibitor of toll-like receptor 4 (TLR4), blocked the cyclophosphamide-induced bladder pain and referred hyperalgesia. Thus, our data indicate involvement of HMGB1 in the cyclophosphamide-induced bladder pain signaling, but not cystitis itself, and suggest that targeting HMGB1 with rhsTM or blocking RAGE might serve as a novel therapeutic strategy for the management of bladder pain. (C) 2013 Elsevier Ltd. All rights reserved.
  • Yan Dong; Miku Hirane; Mutsumi Araki; Nobuyuki Fukushima; Toshifumi Tsujiuchi
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 446 2 585 - 589 2014年04月 [査読有り]
     
    Lysophosphatidic acid (LPA) signaling via G protein-coupled LPA receptors (LPA(1)-LPA(6)) mediates a variety of biological functions, including cell migration. Recently, we have reported that LPA(1) inhibited the cell motile activities of mouse fibroblast 313 cells. In the present study, to evaluate a role of LPA(5) in cellular responses, Lpar5 knockdown (3T3-L5) cells were generated from 3T3 cells. In cell proliferation assays, LPA markedly stimulated the cell proliferation activities of 3T3-L5 cells, compared with control cells. In cell motility assays with Cell Culture Inserts, the cell motile activities of 3T3-L5 cells were significantly higher than those of control cells. The activity levels of matrix metalloproteinases (MMPs) were measured by gelatin zymography. 3T3-L5 cells stimulated the activation of Mmp-2, correlating with the expression levels of Mmp-2 gene. Moreover, to assess the co-effects of LPA(1) and LPA(5) on cell motile activities, Lpar5 knockdown (3T3a1-L5) cells were also established from Lpar1 over-expressing (3T3a1) cells. 3T3a1-L5 cells increased the cell motile activities of 3T3a1 cells, while the cell motile activities of 3T3a1 cells were significantly lower than those of control cells. These results suggest that LPA(5) may act as a negative regulator of cellular responses in mouse fibroblast 313 cells, similar to the case for LPA(1). (C) 2014 Elsevier Inc. All rights reserved.
  • Toshifumi Tsujiuchi; Dai Nakae; Yoichi Konishi
    EXPERIMENTAL AND TOXICOLOGIC PATHOLOGY 66 2-3 81 - 88 2014年03月 [査読有り]
     
    N-Nitrosobis(2-hydroxypropyl)amine (BHP) was first synthesized by Kruger et al. (1974),and has been shown to primarily induce pancreatic duct adenocarcinomas by a subcutaneous injection in Syrian hamsters. By contrast, the carcinogenic effect of BHP has been indicated at the different target organs in rats, namely the lung. When rats are received by an oral administration of BHP in drinking water for 25 weeks, a high incidence of lung carcinomas are induced, which include adenocarcinomas, squamous cell carcinomas and combined squamous cell and adenocarcinomas. So many similarities are observed in terms of not only histological appearances but also gene alterations between human and BHP-induced rat lung cancers. Moreover, the step by step development of lung lesions, from preneoplastic lesions to cancers in rat lung carcinogenesis by BHP offers a good model to investigate the mechanisms underlying the pathogenesis of lung cancers. Because data for genetic and epigenetic alterations have indeed been accumulated during the BHP-induced rat lung carcinogenesis, we will introduce them in this review and hence demonstrate that this lung carcinogenesis model provides a useful opportunity for the research on the pathogenesis of lung cancers of both humans and rats. (C) 2013 Elsevier GmbH. All rights reserved.
  • Toshifumi Tsujiuchi; Mutsumi Araki; Miku Hirane; Yan Dong; Nobuyuki Fukushima
    HISTOLOGY AND HISTOPATHOLOGY 29 3 313 - 321 2014年03月 [査読有り]
     
    Lysophosphatidic acid (LPA) receptors (LPA(1) to LPA(6)) are G protein-coupled transmembrane and mediate a variety of biological responses through the binding of LPA, such as cell proliferation, migration, morphogenesis and differentiation. Previously, high secretion levels of LPA were found in blood and ascites from patients with aggressive ovarian cancer. So far, numerical studies have demonstrated that LPA signaling via LPA receptors contributes to the acquisition of malignant potency by several cancer cells. Moreover, genetic and epigenetic alterations of LPA receptor genes have been detected in cancer cells. Therefore, it is suggested that LPA signaling may be a target molecule for the establishment of chemoprevention agents in clinical cancer approaches. Here, we review the current knowledge for the biological roles of LPA signaling via LPA receptors in the pathogenesis of cancer cells.
  • Serina Inoue; Eriko Tanabe; Ayano Shibata; Miku Hirane; Mutsumi Araki; Yan Dong; Nobuyuki Fukushima; Toshifumi Tsujiuchi
    MOLECULAR AND CELLULAR BIOCHEMISTRY 383 1-2 173 - 177 2013年11月 [査読有り]
     
    Lysophosphatidic acid (LPA) receptors (LPA(1) to LPA(6)) indicate a variety of cellular responses, such as cell proliferation, migration, differentiation, and morphogenesis. However, the role of each LPA receptor is not functionally equivalent. Ethionine, an ethyl analog of methionine, is well known to be one of the potent liver carcinogens in rats. In this study, to assess whether ethionine may regulate cell motile activity through LPA receptors, rat liver epithelial (WB-F344) cells were treated with ethionine for 48 h. In cell motility assay with a cell culture insert, the treatment of ethionine at 1.0 and 10 mu M enhanced significantly high cell motile activity, compared with untreated cells. The expression levels of LPA receptor genes in cells treated with ethionine were measured by quantitative real time RT-PCR analysis. The expression of the Lpar3 gene in ethionine-treated cells was significantly higher than that in untreated cells. Furthermore, to confirm an involvement of LPA(3) on cell motility increased by ethionine, the Lpar3 knockdown cells were also used. The cell motile activity by ethionine was completely suppressed in the Lpar3 knockdown cells. These results suggest that LPA signaling through LPA(3) may be involved in cell motile activity stimulated by ethionine in WB-F344 cells.
  • Miku Hirane; Mutsumi Araki; Yan Dong; Kanya Honoki; Nobuyuki Fukushima; Toshifumi Tsujiuchi
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 441 1 47 - 52 2013年11月 [査読有り]
     
    Reactive oxygen species (ROS) are known to mediate a variety of biological responses, including cell motility. Recently, we indicated that lysophosphatidic acid (LPA) receptor-3 (LPA(3)) increased cell motile activity stimulated by hydrogen peroxide. In the present study, we assessed the role of LPA(1) in the cell motile activity mediated by ROS in mouse fibroblast 3T3 cells. 3T3 cells were treated with hydrogen peroxide and 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) at concentrations of 0.1 and 1 mu M for 48 h. In cell motility assays with Cell Culture Inserts, the cell motile activities of 3T3 cells treated with hydrogen peroxide and DMNQ were significantly higher than those of untreated cells. 3T3 cells treated with hydrogen peroxide and DMNQ showed elevated expression levels of the Lpar3 gene, but not the Lpar1 and Lpar2 genes. To investigate the effects of LPA(1) on the cell motile activity induced by hydrogen peroxide and DMNQ, Lpar1-overexpressing (3T3-a1) cells were generated from 3T3 cells and treated with hydrogen peroxide and DMNQ. The cell motile activities stimulated by hydrogen peroxide and DMNQ were markedly suppressed in 3T3-a1 cells. These results suggest that LPA signaling via LPA(1) inhibits the cell motile activities stimulated by hydrogen peroxide and DMNQ in 3T3 cells. (C) 2013 Elsevier Inc. All rights reserved.
  • 肉腫細胞の浸潤能において、リソフォスファチジル酸シグナルはレセプタータイプ特異的な役割を示す(Receptor type-specific role of lysophosphatidic acid signaling for migration and invasion in sarcoma cells)
    朴木 寛弥; 藤井 宏真; 塚本 真治; 城戸 顕; 森 俊雄; 田中 康仁; 辻内 俊文
    日本癌学会総会記事 72回 323 - 323 2013年10月
  • Eriko Tanabe; Misaho Kitayoshi; Miku Hirane; Mutsumi Araki; Yan Dong; Nobuyuki Fukushima; Toshifumi Tsujiuchi
    Journal of Receptors and Signal Transduction 33 5 286 - 290 2013年10月 [査読有り]
     
    Angiogenesis stimulates the invasive and metastatic process of cancer cells. It is also known that activated fibroblasts promote cancer cell growth and enhance invasive and metastatic potential. Lysophosphatidic acid (LPA) is a biological mediator and interacts with G protein-coupled transmembrane LPA receptors (LPA1 to LPA6). In this study, to assess an involvement of LPA3 on angiogenesis and fibroblast activation, the Lpar3-expressing cells were generated from mouse lung cancer LL/2 cells, which unexpressed LPA3. The Lpar3-expressing cells were maintained in serum-free Dulbecco's modified Eagle's medium for 48h, and cell motility assay was performed with a cell culture Insert. When endothelial F-2 cells and 3T3 fibroblasts were cultured with conditioned medium from the Lpar3-expressing cells, their cell motile activities were significantly lower than the Lpar3-unexpressing (control) cells. Expression levels of vascular endothelial growth factor (Vegf) and fibroblast growth factor (Fgf) genes in the Lpar3-expressing cells were measured by quantitative real time reverse transcription polymerase chain reaction analysis. The expressions of Vegf-A. Fgfa and Fgfb genes in the Lpar3-expressing cells were significantly lower than those in control cells, correlating with the effects on cell motile activities of F-2 and 3T3 cells. These results suggest that LPA signaling through LPA3 may inhibit angiogenesis and fibroblast activation in mouse lung cancer cells. © 2013 Informa Healthcare USA, Inc.
  • Shohei Kuwata; Kei Ohkubo; Shun Kumamoto; Naoto Yamaguchi; Naoki Izuka; Kaeko Murota; Toshifumi Tsujiuchi; Masao Iwamori; Nobuyuki Fukushima
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 439 2 280 - 284 2013年09月 [査読有り]
     
    Lysophosphatidic acid (LPA) is an extracellular lipid mediator consisting of a fatty acid and a phosphate group linked to the glycerol backbone. Here, we show that 1-oleoyl- and 1-palmitoyl-LPA, but not 1-stearoyl- or alkyl-LPA, enhance HNOA ovarian cancer cell survival. Other lysophospholipids with oleic or lauric acid, but not stearic acid, also induce the survival effects. HNOA cells have the lipase activities that cleave LPA to generate fatty acid. Oleic acid stimulates HNOA cell survival via increased glucose utilization. Our findings suggest that extracellular lysolipid metabolism might play an important role in HNOA cell growth. (C) 2013 Elsevier Inc. All rights reserved.
  • Ayano Shibata; Eriko Tanabe; Serina Inoue; Misaho Kitayoshi; Souta Okimoto; Miku Hirane; Mutsumi Araki; Nobuyuki Fukushima; Toshifumi Tsujiuchi
    Biochemical and Biophysical Research Communications 433 3 317 - 321 2013年04月 [査読有り]
     
    Hydrogen peroxide which is one of reactive oxygen species (ROS) mediates a variety of biological responses, including cell proliferation and migration. In the present study, we investigated whether lysophosphatidic acid (LPA) signaling is involved in cell motile activity stimulated by hydrogen peroxide. The rat liver epithelial WB-F344 cells were treated with hydrogen peroxide at 0.1 or 1μM for 48h. In cell motility assays, hydrogen peroxide treated cells showed significantly high cell motile activity, compared with untreated cells. To measure the expression levels of LPA receptor genes, quantitative real time RT-PCR analysis was performed. The expressions of LPA receptor-3 (Lpar3) in hydrogen peroxide treated cells were significantly higher than those in control cells, but not Lpar1 and Lpar2 genes. Next, to assess the effect of LPA3 on cell motile activity, the Lpar3 knockdown cells from WB-F344 cells were also treated with hydrogen peroxide. The cell motile activity of the knockdown cells was not stimulated by hydrogen peroxide. Moreover, in liver cancer cells, hydrogen peroxide significantly activated cell motility of Lpar3-expressing cells, but not Lpar3-unexpressing cells. These results suggest that LPA signaling via LPA3 may be mainly involved in cell motile activity of WB-F344 cells stimulated by hydrogen peroxide. © 2013 Elsevier Inc.
  • Kyoko Okabe; Mai Hayashi; Kohei Kato; Mai Okumura; Rie Fukui; Kanya Honoki; Nobuyuki Fukushima; Toshifumi Tsujiuchi
    MOLECULAR CARCINOGENESIS 52 4 247 - 254 2013年04月 [査読有り]
     
    Lysophosphatidic acid (LPA), which interacts with G protein-coupled transmembrane LPA receptors exhibits several biological effects, such as cell proliferation, migration, and differentiation. Recently, it has been reported that alteration of LPA receptor genes occurs in several cancer cells. In this study, to assess the biological role of LPA receptor-3 (LPA3) in the pathogenesis of tumor cells, we generated the Lpar3-expressing cells (RHa3B12 and RHa3G8) from rat hepatoma RH7777 cells, and examined their abilities of cell migration and tumorigenicity, compared with the Lpar3-unexpressing cells. In cell motility and invasion assays, RHa3B12 and RHa3G8 cells showed significantly higher intrinsic activity without LPA treatment than control RH7777AB cells. LPA treatment further increased cell motility and invasion of these cells. The cell motility of RHa3B12 and RHa3G8 cells stimulated by LPA treatment was significantly suppressed by pretreatment with inhibitors of Gi or Gq proteins. In a soft agar assay, the large sized colonies were formed in RHa3B12 and RHa3G8 cells, but not in RH7777AB cells. The cell survival of RHa3G8 cells treated with cisplatin (CDDP) or doxorubicin (DOX) was higher than that of RH7777AB cells, correlating with the elevated expression levels of multidrug-resistance related genes, Mdr1a, Mdr1b, and Gstp1. These results suggest that LPA3 may be involved in progression and aggressiveness of rat hepatoma RH7777 cells. (c) 2011 Wiley Periodicals, Inc.
  • Kyohei Yoshikawa; Eriko Tanabe; Ayano Shibata; Serina Inoue; Misaho Kitayoshi; Souta Okimoto; Nobuyuki Fukushima; Toshifumi Tsujiuchi
    EXPERIMENTAL CELL RESEARCH 319 3 105 - 112 2013年02月 [査読有り]
     
    Lysophosphatidic acid (LPA) mediates a variety of cellular responses with atleast six G protein-coupled transmembrane receptors (LPA receptor-1 (LPA(1)-LPA(6))). The interaction between LPA receptors and other cellular molecules on the biological function is not fully understood. Recently, we have reported that LPA(1) suppressed and LPA(3) stimulated cell migration of pancreatic cancer cells. In the present study, to evaluate the function of LPA(2) on motile and invasive activities of pancreatic cancer cells, we generated Lpar2 knockdown (HPD-sh2) cells from hamster pancreatic cancer cells and measured their cell migration ability. In cell motility and invasive assays with an uncoated Cell Culture Insert, HPD-sh2 cells showed significantly lower intrinsic activity than control (HPD-GFP) cells. Since K-ras mutations were frequently detected in pancreatic cancer, we next investigated whether oncogenic K-ras is involved in cell migration induced by LPA2 using K-ras knockdown (HPD-K2) cells. The cell motile ability of HPD-K2 cells was significantly lower than that of control cells. To confirm LPA2 increases cell migration activity, cells were pretreated with dioctylglycerol pyrophosphate (DGPP) which is the antagonist of LPA(1)/LPA(3). The cell motile and invasive abilities of DGPP -treated HPD-GFP cells were markedly higher than those of untreated cells, but DGPP did not stimulate cell migration of HPD-K2 cells. These results suggest that cell migration activity of pancreatic cancer cells stimulated by LPA2 may be enhanced by oncogenic K-ras. (c) 2012 Elsevier Inc. All rights reserved.
  • Eriko Tanabe; Misaho Kitayoshi; Kyohei Yoshikawa; Ayano Shibata; Kanya Honoki; Nobuyuki Fukushima; Toshifumi Tsujiuchi
    JOURNAL OF RECEPTORS AND SIGNAL TRANSDUCTION 32 6 328 - 334 2012年12月 [査読有り]
     
    Lysophosphatidic acid (LPA) interacts with at least six G protein-coupled transmembrane LPA receptors (LPA(1)-LPA(6)). Recently, we have reported that LPA(3) indicated opposite effects on cell migration, depending on the cell types. In the present study, to assess an involvement of LPA(3) on cell migration of sarcoma cells, we generated LPA receptor-3 (LPAR3)-knockdown (HT1080-sh3 and HOS-sh3, respectively) cells from fibrosarcoma HT1080 and osteosarcoma HOS cells, and measured their cell migration abilities. In cell motility assay with a Cell Culture Insert, both LPAR3-knockdown cells showed significantly lower cell motile activities than control cells. Next, to investigate the effect of LPAR3-knockdown on invasion activity, which degraded the extracellular matrices, the Matrigel-coated filter was used. HT1080-sh3 cells showed significantly low invasive activity compared with control cells, while no invasive activity was found in HOS-sh3 cells. In gelatin zymography, no significant difference of matrix metalloproteinase (MMP)-2 and MMP-9 activities were detected in all cells. The results indicated that LPA(3) acts as a positive regulator of cell motility and invasion in sarcoma cells, suggesting that LPA signaling pathway via LPA(3) may be involved in the progression of sarcoma cells.
  • Rie Fukui; Eriko Tanabe; Misaho Kitayoshi; Kyohei Yoshikawa; Nobuyuki Fukushima; Toshifumi Tsujiuchi
    TUMOR BIOLOGY 33 6 1899 - 1905 2012年12月 [査読有り]
     
    Lysophosphatidic acid (LPA) mediates a wide range of biological responses with G protein-coupled transmembrane receptors (LPA receptors). So far, at least six types of LPA receptors (LPA receptor-1 (LPA(1)) to LPA(6)) have been identified. Recently, it has been reported that LPA(3) indicates opposite effects on cellular functions of cancer cells. In the present study, to assess a biological role of LPA(3) on cell migration ability of colon cancer cells, we generated LPA receptor-3 (LPAR3) knockdown (HCT-sh3-3) cells from HCT116 and measured cell motile and invasion activities. In motility assay with a cell culture insert, HCT-sh3-3 cells showed significantly high cell motile activity, compared with control cells. For invasion assay, the filter was coated with Matrigel. The invasive activity of HCT-sh3-3 cells was significantly higher than that of control cells. Furthermore, we also examined the effects of LPAR3 knockdown on the interaction between colon cancer cells and endothelial F-2 cells. When F-2 cells were cultured with serum-free DMEM containing a supernatant from HCT-sh3-3 cells, the cell growth rate and migration activity of F-2 cells were significantly stimulated, associating with the elevated expressions of vascular endothelial growth factor (VEGF)-A and VEGF-C genes in HCT-sh3-3 cells. These results suggest that LPA(3) may act as a negative regulator on cell motile and invasive abilities of colon cancer HCT116 cells.
  • Eriko Tanabe; Ayano Shibata; Serina Inoue; Misaho Kitayoshi; Nobuyuki Fukushima; Toshifumi Tsujiuchi
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 428 1 105 - 109 2012年11月 [査読有り]
     
    Lysophosphatidic acid (LPA) interacts with G protein-coupled transmembrane LPA receptors (LPA receptors; LPA(1)-LPA(6)). Recently, we demonstrated that each LPA receptor acts as a positive or negative regulator of cell migration ability. It is known that estrogens indicate a variety of biological functions, including cell motility. In the present study, to assess whether LPA signaling is involved in cell motile activity stimulated by estrogens, we measured cell motile activity and LPA receptor expressions of rat liver epithelial WB-F344 cells treated with 17 beta-estradiol (E-2), ethinyl estradiol (EE) and diethylstilbestrol (DES) at concentrations of 0.1 and 1.0 mu M for 48 h. The cell motility of E-2 and EE treated cells was significantly higher than that of untreated cells. By contrast, DES markedly inhibited cell motile activity. Using quantitative real time RT-PCR analysis, Lpar1 and Lpar3 expressions in E-2 treated cells were significantly higher than those in untreated cells. In EE treated cells, Lpar3 expression was markedly elevated, whereas Lpar1 expression was decreased. On the other hand, Lpar1 expression was significantly increased in DES treated cells. Interestingly, the effects of E-2, EE and DES on cell motility were suppressed by Lpar1 or Lpar3 knockdown. These results suggest that the different induction of LPA receptors by estrogens may regulate cell motile activity of WB-F344 cells. (C) 2012 Elsevier Inc. All rights reserved.
  • Maho Matsunami; Takahiro Miki; Kanae Nishiura; Yuko Hayashi; Yasumasa Okawa; Hiroyuki Nishikawa; Fumiko Sekiguchi; Lisa Kubo; Tomoka Ozaki; Toshifumi Tsujiuchi; Atsufumi Kawabata
    BRITISH JOURNAL OF PHARMACOLOGY 167 4 917 - 928 2012年10月 [査読有り]
     
    BACKGROUND AND PURPOSE Hydrogen sulfide (H2S), generated by enzymes such as cystathionine-?-lyase (CSE) from L-cysteine, facilitates pain signals by activating the Cav3.2 T-type Ca2+ channels. Here, we assessed the involvement of the CSE/H2S/Cav3.2 pathway in cystitis-related bladder pain. EXPERIMENTAL APPROACH Cystitis was induced by i.p. administration of cyclophosphamide in mice. Bladder pain-like nociceptive behaviour was observed and referred hyperalgesia was evaluated using von Frey filaments. Phosphorylation of ERK in the spinal dorsal horn was determined immunohistochemically following intravesical administration of NaHS, an H2S donor. KEY RESULTS Cyclophosphamide caused cystitis-related symptoms including increased bladder weight, accompanied by nociceptive changes (bladder pain-like nociceptive behaviour and referred hyperalgesia). Pretreatment with DL-propargylglycine, an inhibitor of CSE, abolished the nociceptive changes and partly prevented the increased bladder weight. CSE protein in the bladder was markedly up-regulated during development of cystitis. Mibefradil or NNC 550396, blockers of T-type Ca2+ channels, administered after the symptoms of cystitis appeared, reversed the nociceptive changes. Further, silencing of Cav3.2 protein by repeated intrathecal administration of mouse Cav3.2-targeting antisense oligodeoxynucleotides also significantly attenuated the nociceptive changes, but not the increased bladder weight. Finally, the number of cells staining positive for phospho-ERK was increased in the superficial layer of the L6 spinal cord after intravesical administration of NaHS, an effect inhibited by NNC 550396. CONCLUSION AND IMPLICATIONS Endogenous H2S, generated by up-regulated CSE, caused bladder pain and referred hyperalgesia through the activation of Cav3.2 channels, one of the T-type Ca2+ channels, in mice with cyclophosphamide-induced cystitis.
  • Kohei Kato; Kyohei Yoshikawa; Eriko Tanabe; Misaho Kitayoshi; Rie Fukui; Nobuyuki Fukushima; Toshifumi Tsujiuchi
    TUMOR BIOLOGY 33 5 1739 - 1744 2012年10月 [査読有り]
     
    Lysophosphatidic acid (LPA) interacts with at least six G protein-coupled transmembrane LPA receptors. Recently, it has been demonstrated that each LPA receptor acts as a positive or negative regulator of cellular function. In the present study, to assess a biological role of LPA receptors on cell migration of pancreatic cancer cells, we generated LPA receptor-1 (LPA(1)) and LPA(3) knockdown cells from hamster pancreatic cancer cells by transfection with short hairpin RNA plasmids and measured their cell motile and invasive abilities. In cell motility and invasion assay, a Cell Culture Insert, coated with or without a Matrigel, was used. While the cell motility and invasion of Lpar1 knockdown cells were markedly enhanced than those of control cells, Lpar3 knockdown cells showed significantly lower cell motility and invasion. Moreover, to investigate an involvement of LPA(1) and LPA(3) in the development of pancreatic cancers, we also measured the expression levels of Lpar1 and Lpar3 genes in hamster pancreatic duct adenocarcinomas (PDAs) induced by a nitroso compound. The expressions of Lpar1 gene in PDAs were significantly lower than those in normal pancreatic tissues. By contrast, the elevated expressions of Lpar3 gene were detected in PDAs. We thus demonstrate that LPA(1) and LPA(3) play the different roles on cell migration ability of pancreatic cancer cells, suggesting the opposite effects via LPA(1) and LPA(3) may contribute to the pathogenesis of pancreatic cancers in hamsters.
  • リゾホスファチジン酸受容体阻害はヒト肉腫細胞の運動・浸潤能を抑制する(Inhibition of lysophosphatidic acid receptor-3 suppresses cell migration and invasion activity in human sarcoma cells)
    朴木 寛弥; 藤井 宏真; 城戸 顕; 塚本 真治; 辻内 俊文
    日本癌学会総会記事 71回 422 - 422 2012年08月
  • Misaho Kitayoshi; Rie Fukui; Eriko Tanabe; Kohei Kato; Kyohei Yoshikawa; Nobuyuki Fukushima; Toshifumi Tsujiuchi
    JOURNAL OF RECEPTORS AND SIGNAL TRANSDUCTION 32 4 209 - 213 2012年08月 [査読有り]
     
    Lysophosphatidic acid (LPA) receptors belong to G protein-coupled transmembrane receptors and mediate a variety of cellular responses through the binding of LPA. So far, six types of LPA receptors (LPA receptor-1 (LPA(1)) to LPA(6)) have been identified. Recently, it has been demonstrated that each LPA receptor has opposite effects on malignant property of cancer cells. In this study, to evaluate an involvement of LPA receptors on angiogenic process in mammary tumor cells, we generated Lpar1- and Lpar3-expressing (FM3A-a1 and FM3A-a3A9, respectively) cells from FM3A cells, and investigated the effects on cell proliferation and migration abilities of endothelial F-2 cells by those cells. In Vegf-A and Vegf-C genes, FM3A-a1 cells indicated high expression and FM3A-a3A9 cells showed low expression, compared with control cells. When F-2 cells were cultured with a supernatant from FM3A-a1 cells, the cell growth rate and migration ability of F-2 cells was significantly higher than control cells. By contrast, a supernatant from FM3A-a3A9 cells significantly inhibited those abilities of F-2 cells. These results suggest that LPA(1) and LPA(3) may play opposite roles on the regulation of endothelial cells in mouse mammary tumor FM3A cells.
  • Misaho Kitayoshi; Kohei Kato; Eriko Tanabe; Kyohei Yoshikawa; Rie Fukui; Nobuyuki Fukushima; Toshifumi Tsujiuchi
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 422 2 339 - 343 2012年06月 [査読有り]
     
    Lysophosphatidic acid (LPA) receptors belong to G protein-coupled transmembrane receptors (LPA receptors; LPA(1) to LPA(6)). They indicate a variety of cellular response by the interaction with LPA, including cell proliferation, migration and differentiation. Recently, we have reported that constitutive active mutated LPA(1) induced the strong biological effects of rat neuroblastoma B103 cells. In the present study, we examined the effects of mutated LPA(1) on the interaction between B103 cells and endothelial F-2 cells. Each LPA receptor expressing B103 cells were maintained in serum-free DMEM and cell motility assay was performed with a Cell Culture Insert. When F-2 cells were cultured with conditioned medium from Lpar1 and Lpar3-expressing cells, the cell motility of F-2 cells was significantly higher than control cells. Interestingly, the motile activity of F-2 cells was strongly induced by mutated LPA(1) than other cells, correlating with the expression levels of vascular endothelial growth factor (Vegf)-A and Vegf-C. Pretreatment of LPA signaling inhibitors inhibited F-2 cell motility stimulated by mutated LPA(1). These results suggest that activation of LPA signaling via mutated LPA(1) may play an important role in the promotion of angiogenesis in rat neuroblastoma cells. (C) 2012 Elsevier Inc. All rights reserved.
  • Daisuke Furuta; Masayuki Yamane; Toshifumi Tsujiuchi; Ryutaro Moriyama; Nobuyuki Fukushima
    MOLECULAR AND CELLULAR NEUROSCIENCE 50 1 21 - 34 2012年05月 [査読有り]
     
    Although neurite branching is crucial for neuronal network formation after birth, its underlying mechanisms remain unclear. Here, we demonstrate that lysophosphatidic acid (LPA) stimulates neurite branching through a novel signaling pathway. Treatment of neuronal cell lines with LPA resulted in neurite branch formation when LPA(3) receptor was introduced. The effects of LPA were blocked by inhibition of G(q) signaling. Furthermore, expression of inhibitory mutants of the small GTPase Rnd2/Rho7 or an Rnd2 effector rapostlin abolished LPA(3)-mediated neurite branching. The LPA(3) agonist 2(S)-OMPT or LPA also induced axonal branch formation in hippocampal neurons, which was blocked by G(q) and Rnd2 pathway inhibition or LPA(3) knockdown. These findings suggest that the novel signaling pathway involving LPA(3), G(q), and Rnd2 may play an important role in neuronal network formation. (c) 2012 Elsevier Inc. All rights reserved.
  • Mai Hayashi; Kyoko Okabe; Kohei Kato; Mai Okumura; Rie Fukui; Nobuyuki Fukushima; Toshifumi Tsujiuchi
    CANCER LETTERS 316 1 91 - 96 2012年03月 [査読有り]
     
    Lysophosphatidic acid (LPA) is a bioactive lipid mediator that induces diverse cellular biological effects and interacts with G protein-coupled transmembrane LPA receptors. In the present study, to assess biological roles of LPA receptors in the pathogenesis of tumor cells, each LPA receptor (Lpar1, Lpar2 or Lpar3)-expressing rat neuroblastoma B103 cells (Ipa1-1, Ipa2-2 or Ipa3-3-2 cells, respectively) were used. In cell motility and invasion assay, Ipa2-2 and Ipa3-3-2 cells showed significant higher intrinsic activity without LPA treatment than LPA receptor-unexpressing AB2-1 bf cells. LPA treatment further increased cell motility of these cells, which was suppressed by the pretreatment with inhibitors of Gi, Gq protein, or ROCK. By contrast, Ipa1-1 cells markedly decreased intrinsic cell motility and invasion, compared with AB2-1 bf cells. Constitutively active mutant Lpar1-expressing cells (Ipa1 Delta-1) showed significant high motility, comparable with those of Ipa2-2 and Ipa3-3-2. In soft agar assay. Ipa3-3-2 and Ipa1 Delta-1 cells showed colony formation, but other cells failed. These results suggest that LPA receptors may play different roles in cell proliferation and migration of rat neuroblastoma cells. (C) 2011 Elsevier Ireland Ltd. All rights reserved.
  • Tsukasa Kitahashi; Shoko Ikawa; Akika Sakamoto; Yoshihiro Nomura; Toshifumi Tsujiuchi; Kenji Shimizu; Shuji Sasabe; Eun Young Park; Yasushi Nakamura; Masahiro Tsutsumi; Kenji Sato
    JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY 60 4 940 - 945 2012年02月 [査読有り]
     
    A water extract of shark cartilage was fractionated into acidic and basic fractions by preparative isoelectric focusing on the basis of the amphoteric nature of samples. The acidic fraction was further fractionated into ethanol-soluble and precipitate fractions. After the carcinogenesis treatment using N-nitrosobis(2-oxopropyl)amine, hamsters received a diet containing each fraction or purified chondroichin sulfate to give 0.4% (w/w) for 50 days. Only administration of the acidic ethanol-precipitate-fraction-containing diet significantly increased serum inhibitory activity against matrix metalloproteinase (MMP)-9 and reduced the number of adenocarcinomas in the pancreatic duct. The active fraction predominantly consisted of chondroichin sulfate-containing proteoglycan. However, the purified chondroichin sulfate had no significant activity. These results suggest that the protein moiety of the proteoglycan might be involved in the increase of serum inhibitory activity against MMP-9 and suppression of pancreatic carcinogenesis in hamster.
  • Kohei Kato; Rie Fukui; Kyoko Okabe; Eriko Tanabe; Misaho Kitayoshi; Nobuyuki Fukushima; Toshifumi Tsujiuchi
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 417 2 790 - 793 2012年01月 [査読有り]
     
    Lysophosphatidic acid (LPA) is a simple phospholipid which interacts with at least six G protein-coupled transmembrane LPA receptors (LPA(1)-LPA(6)). In rat neuroblastoma B103 cells, we have recently reported that each LPA receptor indicates the different cellular functions, including cell motility, invasion and tumorigenicity. Especially, mutated and constitutively active LPA(1) enhanced these cellular effects in B103 cells. In the present study, to better understand a role of mutated LPA(1) underlying progression of cancer cells, we measured the expression and activity levels of matrix metalloproteinases (MMPs) in constitutively active mutant Lpar1-expressing B103 cells (lpa1 Delta-1), compared with each wild-type LPA receptor-expressing cells. LPA receptor-unexpressing cells were also used as control. In quantitative real time RT-PCR analysis, the expressions of Mmp-9 were detected at the same levels in all cells. By contrast, Mmp-2 expressions of lpa1 Delta-1 were significantly higher than those of other cells. In gelatin zymography, proMmp-9 was observed at the same levels in all cells. Interestingly, markedly high levels of proMmp-2 and Mmp-2 were detected in lpa1 Delta-1 cells, whereas no activation was in other cells. The increased expression and activity of Mmp-2 in lpa1 Delta-1 cells were suppressed by the pretreatment with a Gq protein inhibitor. These results suggest that mutated LPA(1) may involve in the enhancement of Mmp-2 expression and activation in rat neuroblastoma cells. (C) 2011 Elsevier Inc. All rights reserved.
  • Rie Fukui; Kohei Kato; Kyoko Okabe; Misaho Kitayoshi; Eriko Tanabe; Nobuyuki Fukushima; Toshifumi Tsujiuchi
    Journal of Toxicologic Pathology 25 3 225 - 228 2012年 [査読有り]
     
    Lysophosphatidic acid (LPA) acts as a simple phospholipid that interacts with G protein-coupled transmembrane LPA recep- tors. Recently, it has been reported that each LPA receptor plays different biological roles in acquisition of the malignant property of tumor cells. In this study, to assess the involvement of LPA receptor-3 (LPA 3) in cell survival after treatment with anticancer drugs, we generated Lpar3-expressing FM3A-a3A9 cells from mouse mammary tumor FM3A cells and examined the cell survival rate after treatment with anticancer drugs compared with Lpar3-unexpressing cells. Cells were treated with 0.005 to 10 μM of cisplatin (CDDP) or doxorubicin (DOX) for 3 days. For the CDDP and DOX treatments, the cell survival rate of FM3A-a3A9 cells was significantly higher than that of Lpar3-unexpressing cells. The expression level of the Mdrla gene in FM3A-a3A9 cells was higher than that of Lpar3-unexpressing cells, whereas no significant difference in multidrug resistance 1b (Mdr1b) and glutathione S-transferase mu1 (Gstm1) expressions was found. These results suggest that LPA 3 may enhance the cell survival rate after treatment with anticancer drugs in mouse mammary tumor cells, correlating with increased expression of the Mdr1 gene. © 2012 The Japanese Society of Toxicologic Pathology.
  • Shinji Tsukamoto; Kanya Honoki; Hiromasa Fujii; Yasuaki Tohma; Akira Kido; Toshio Mori; Toshifumi Tsujiuchi; Yasuhito Tanaka
    INTERNATIONAL JOURNAL OF ONCOLOGY 40 1 163 - 169 2012年01月 [査読有り]
     
    Although mesenchymal stern cells (MSCs) are considered to be the cells of origin for most sarcomas, the role of MSCs as a source of tumor stroma is not fully understood in this tumor type. The current study investigated whether MSCs affect the tumor growth and metastatic ability in rat osteosarcoma model. Results from subcutaneous co-implantation of rat osteosarcoma COSINR cells, established in our laboratory, with rat MSCs isolated from femur bone marrow showed that the incidence of tumor formation and tumor growth rate was higher until 5 weeks compared to COSINR cell inoculation alone. However, no difference was observed in tumor growth afterwards and in the number of metastatic nodules at 9 weeks (0.75 vs. 1.2). Intravenous MSC injection at weeks 3 and 5 after subcutaneous inoculation of COSINR cells significantly increased the number of lung nodules in the group with MSC injection compared to the group without MSC injection (17.33 vs. 2.0), while no difference was observed in subcutaneous tumor growth between those groups. Pathway analysis from gene expression profile identified that genes involved in focal adhesion, cytokine-cytokine receptor and extracellular matrix-receptor pathways such as CAMs (ICAM and VCAM)-integrins were highly expressed in MSCs, possibly participating in the tumor progression of osteosarcoma. These results suggest that MSCs could provide a source of microenvironments for osteosarcoma cells, and might enhance the ability of settlement and colonization which lead to early onset of growth and metastasis, possibly through their activated pathways interaction.
  • Comparison of gene expression profiling in sarcoma and mesenchymal stem cells identifies tumorigenic pathways in chemically induced rat sarcoma model.
    Honoki K; Fujii H; Tohma Y; Tsujiuchi T; Kido A; Tsukamoto S; Mori T; Tanaka Y
    ISRN Oncol 2012年 [査読有り]
  • Mai Okumura; Kohei Kato; Rie Fukui; Nobuyuki Fukushima; Toshifumi Tsujiuchi
    Journal of Toxicologic Pathology 24 3 183 - 186 2011年10月 [査読有り]
     
    The tumor promoting agent 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulates cell migration of several tumor cells. Recently, we reported that loss of lysophosphatidic acid (LPA) receptor-3 (LPA3) enhanced cell migration of murine lung tumor LL/2 cells. In the present study, we investigated whether LPA3 is involved in cell migration of mouse lung tumor cells stimulated by TPA. Exogenous LPA3 gene (Lpar3)-expressing (LL/2-a3) cells and LL/2-AB cells as a vector control generated from LL/2 cells were used. In a cell migration assay, TPA treatment significantly stimulated cell migration of LL/2-AB and LL/2-a3 cells, while the cell migration abilities of LL/2-a3 were markedly lower than those of LL/2-AB cells. Using quantitative real-time reverse transcription (RT)-polymerase chain reaction (PCR) analysis, no effect of TPA treatment on the expression levels of LPA1, LPA2 and LPA3 genes was detected in either type of cells. These results suggest that the LPA3 may not be involved in the enhanced migration ability by TPA in mouse lung tumor cells. ©2011 The Japanese Society of Toxicologic Pathology. © 2011 The Japanese Society of Toxicologic Pathology.
  • Toshifumi Tsujiuchi; Kyoko Okabe; Nobuyuki Fukushima
    Journal of Toxicologic Pathology 24 3 143 - 148 2011年10月 [査読有り]
     
    Lysophosphatidic acid (LPA) is a bioactive mediator and induces several biological effects, including cell proliferation, migration, morphogenesis and differentiation. LPA interacts with at least six G protein-coupled receptors (GPCRs), including LPA receptor-1 (LPA 1), LPA 2, LPA 3, LPA 4, LPA 5 and LPA 6. These receptors show different biological functions through the binding of LPA, depending on the type of cells. In human malignancies, a high level of LPA production was found in plasma and ascites in ovarian cancer cases. Moreover, aberrant expression levels of LPA receptor genes were detected in some cancer cells. Therefore, it is suggested that LPA receptors may be involved in the pathogenesis of tumor cells as well as LPA per se. Recently, we have reported that alterations of LPA receptor genes also occur in rodent tumors. In this review, we summarize the recent evidence in the investigations of LPA receptor alterations in rodent tumors by experimental models. © 2011 The Japanese Society of Toxicologic Pathology.
  • Kyoko Okabe; Mai Hayashi; Ikuma Yoshida; Kazuki Nishimura; Nobuyuki Fukushima; Toshifumi Tsujiuchi
    ARCHIVES OF TOXICOLOGY 85 10 1303 - 1310 2011年10月 [査読有り]
     
    Altered expressions of lysophosphatidic acid (LPA) receptor genes have been reported in tumor cells of human and rats. Recently, we detected the frequent mutations of LPA receptor-1 (LPA1) gene in rat hepatocellular carcinomas (HCCs) induced by a choline-deficient L-amino acid-defined (CDAA) diet. In this study, the DNA methylation patterns of LPA receptor genes and their expression levels during rat hepatocarcinogenesis induced by the CDAA diet were investigated. Six-week-old F344 male rats were continuously fed with the CDAA diet, and animals were then killed at 7 days and 2, 12, 20, and 75 weeks, respectively. Genomic DNAs were extracted from livers and HCCs for the assessment of methylation status by bisulfite sequencing, comparing to normal livers. The livers of rats fed the CDAA diet were unmethylated in LPA1 and LPA2 genes as well as normal livers. In LPA3 gene, although normal livers were unmethylated, the livers at 7 days and 2 and 12 weeks weakly or moderately methylated and those at 20 weeks markedly methylated. Moreover, 4 HCCs were completely methylated in LPA3 gene. Expression levels of LPA receptor genes in the livers of rats fed the CDAA diet and HCCs were correlating with DNA methylation status. These results indicate that DNA methylation status of the LPA3 gene was disturbed in the livers of rats fed the CDAA diet and established HCCs, suggesting that alterations of the LPA receptor genes might be involved during rat hepatocarcinogenesis induced by the CDAA diet.
  • Kyoko Okabe; Kohei Kato; Miki Teranishi; Mai Okumura; Rie Fukui; Toshio Mori; Nobuyuki Fukushima; Toshifumi Tsujiuchi
    CANCER LETTERS 309 2 236 - 242 2011年10月 [査読有り]
     
    12-O-tetradecanoylphorbol-13-acetate (TPA) which is one of tumor promoting agents stimulates cell migration ability of several tumor cells. In the present study, we investigated whether lysophosphatidic acid (LPA) receptors are involved in cell migration of rat liver cells stimulated by TPA. The rat liver epithelial WB-F344 and hepatoma RH7777 cells were treated by TPA for 48 h. The expression levels of LPA receptor genes in those cells were measured by real-time reverse transcription (RT)-polymerase chain reaction (PCR) analysis. The expressions of the LPA receptor-3 (Lpar3) gene were significantly elevated in WB-F344 and RH7777 cells treated by TPA, but not Lpar1 and Lpar2 genes. In cell migration assay, TPA treatment showed markedly high cell migration in both cells. The pretreatment with inhibitors of Gi protein suppressed those migration abilities. We next generated the Lpar3 knockdown cells from WB-F344 cells and investigated the effect on cell migration. Interestingly, the cell migration of the knockdown cells was not stimulated by TPA. These results suggest that TPA-stimulated cell migration of rat liver cells may be mainly dependent on the LPA(3)-mediated effect. (C) 2011 Elsevier Ireland Ltd. All rights reserved.
  • Kyoko Okabe; Mai Hayashi; Yasuna Yamawaki; Miki Teranishi; Kanya Honoki; Toshio Mori; Nobuyuki Fukushima; Toshifumi Tsujiuchi
    MOLECULAR CARCINOGENESIS 50 8 635 - 642 2011年08月 [査読有り]
     
    Aberrant expressions of lysophosphatidic acid (LPA) receptor genes have been reported in tumor cells. Here, we measured the expression levels of the Lpa5 gene and its DNA methylation status in rat tumor cells, and investigated cell growth effects of LPA in Lpa5 expressed cells. Real-time reverse transcription (RT)-polymerase chain reaction (PCR) analysis revealed that increased expressions of the Lpa5 gene were detected in rat liver-derived hepatoma RH7777 and lung-derived adenocarcinoma RLCNR cells. For the analysis of methylation status, bisulfite sequencing was performed with RH7777 and RLCNR cells and compared with other tumor cells and liver epithelial cells. The Lpa5 gene in Lpa5 unexpressed cells and liver epithelial cells were highly methylated in the 50 upstream region. In contrast, the Lpa5 gene in RH7777 and RLCNR cells was unmethylated, correlating with increased expressions of Lpa5. In the assays for cell growth effects of LPA, LPA enhanced cell proliferation and motility in RH7777 and RLCNR cells. LPA also stimulated cell invasion in RLCNR, but not in RH7777 cells. In rat liver and lung tumors induced by nitroso-compounds, 4 out of 6 hepatocellular carcinomas and 5 out of 6 lung adenocarcinomas indicated increased expressions of Lpa5 with unmethylated status. These results suggest that increased Lpa5 expressions due to aberrant DNA methylation may be involved in the acquisition of growth advantage of rat tumor cells. (C) 2011 Wiley-Liss, Inc.
  • Nobuyuki Fukushima; Daisuke Furuta; Toshifumi Tsujiuchi
    NEUROCHEMISTRY INTERNATIONAL 59 2 109 - 113 2011年08月 [査読有り]
     
    Neurite development requires rearrangement of cytoskeletal elements, which are mechanically and functionally integrated with each other. Although the process of how an extracellular signal induces rearrangement of a single element has been closely examined, the mechanisms by which the signal regulates cytoskeletal integration during cell shape changes are poorly understood. We previously reported that lysophosphatidic acid (LPA) induces actin polymerization-dependent microtubule (MT) rearrangement, leading to neurite retraction in cultured neurons. Here we examined whether the crosslinker proteins were involved in LPA-induced neurite retraction using immortalized mouse neuroblast TR cells. When the MT-binding domains of MACF (MT actin-crosslinking factor) were exogenously expressed in TR cells, MTs were found to be stabilized and become resistant to exposure to LPA. On the other hand, expression of MT-associated protein 2c showed no effect on LPA-induced neurite retraction. These findings suggest that MACF is involved in actin-dependent MT rearrangement during LPA-induced neurite retraction. (C) 2011 Elsevier BM. All rights reserved.
  • Naoko Wakabayashi; Kyoko Okabe; Mai Hayashi; Kanya Honoki; Toshifumi Tsujiuchi
    ANTICANCER RESEARCH 31 2 543 - 547 2011年02月 [査読有り]
     
    To clarify the involvement of the Stk11/Lkb1 gene in the development of hepatocellular carcinomas (HCCs), its alteration in rat HCCs induced by exogenous and endogenous liver carcinogenesis models was investigated. Materials and Methods: Fifteen HCCs induced by N-nitrosodiethylamine (DEN) and 12 HCCs induced by a choline-deficient L-amino acid-defined (CDAA) diet were obtained. To assess mutations and aberrant transcriptions of the Stk11 gene, polymerase chain reaction (PCR) single strand conformation polymorphism (SSCP) and reverse transcription (RT)-PCR analyses were performed, respectively. Results: A mutation was detected in only 1 out of 15 HCCs by DEN, but no mutations in 12 HCCs by the CDAA diet. Aberrant transcripts were found in 4 out of 15 HCCs by DEN and in 3 out of 12 HCCs by the CDAA diet. Conclusion: These results suggest that alterations of the Stk11 gene may play a limited role in both exogenous and endogenous rat liver carcinogenesis.
  • Mai Hayashi; Kyoko Okabe; Yasuna Yamawaki; Miki Teranishi; Kanya Honoki; Toshio Mori; Nobuyuki Fukushima; Toshifumi Tsujiuchi
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 405 3 450 - 454 2011年02月 [査読有り]
     
    Lysophosphatidic acid (LPA) indicates several biological effects, such as cell proliferation, differentiation and migration. LPA interacts with G protein-coupled transmembrane LPA receptors. In our previous report, we detected that loss of the LPA receptor-1 (Lpar1) expression is due to its aberrant DNA methylation in rat tumor cell lines. In this study, to assess an involvement of the other LPA receptor, Lpar3, in the pathogenesis of rat lung tumor cells, we measured the expression levels of the Lpar3 gene and its DNA methylation status by reverse transcription (RT)-polymerase chain reaction (PCR) and bisulfite sequencing analyses, respectively. RLCNR lung adenocarcinoma cells showed reduced expression of the Lpar3, compared with normal lung tissues. In the 5' upstream region of the Lpar3, normal lung tissues were unmethylated. By contrast, RLCNR cells were highly methylated, correlating with reduced expressions of the Lpar3. Based on these results, we generated the Lpar3-expressing RLCNR-a3 cells and measured the cell migration ability. Interestingly, the cell migration of RLCNR-a3 cells was significantly lower than that of RLCNR cells. This study suggests that loss of the Lpar3 due to aberrant DNA methylation may be involved in the progression of rat lung tumor cells. (C) 2011 Elsevier Inc. All rights reserved.
  • Megumu Tsujino; Minako Fujii; Kyoko Okabe; Toshio Mori; Nobuyuki Fukushima; Toshifumi Tsujiuchi
    VIRCHOWS ARCHIV 457 6 669 - 676 2010年12月 [査読有り]
     
    Lysophosphatidic acid (LPA), which is a bioactive phospholipid, interacts with specific G protein-coupled transmembrane receptors. Recently, alterations of LPA receptor genes have been reported in some tumor cells. In this study, we examined the expression profiles and DNA methylation status of LPA receptor 1-5 (LPA1-5) genes in human colon cancer cells and also looked for the mutations. Reverse transcription-polymerase chain reaction (PCR) and bisulfite sequencing analyses were carried out. While LPA1, LPA2, and LPA4 genes were expressed in DLD1, SW480, HCT116, CaCo-2, SW48, and LoVo cells, the expressions of LPA3 and LPA5 genes were various. These expression levels were correlated with DNA methylation status in the 5' upstream regions of the LPA receptor genes. Mutation analysis was also performed using a PCR-single-strand conformation polymorphism method. Although no mutations in LPA1, LPA3 and LPA5 genes were found in all types of cells, LPA2 mutations in DLD1 and SW48 cells, and LPA4 mutation were found in DLD1 cells. On the basis of the present results, we demonstrate that these colon cancer cells will be available to understanding the molecular pathway through LPA receptors in the development of tumor cells, and that LPA receptors may be new molecular targets for therapeutic approaches and chemoprevention.
  • Kanya Honoki; Hiromasa Fujii; Atsushi Kubo; Akira Kido; Toshio Mori; Yasuhito Tanaka; Toshifumi Tsujiuchi
    ONCOLOGY REPORTS 24 2 501 - 505 2010年08月 [査読有り]
     
    Elevated aldehyde dehydrogenase 1 (ALDH1) has been proposed as one of the possible candidates for a stem cell maker that can be used for the isolation of cancer stem cells from cancers such as leukemia and breast cancer. In the current study, we found that subpopulations with elevated ALDH1 were present in the human sarcoma cell lines, MG63 (osteosarcoma) and HT 1080 (fibrosarcoma), using Aldefluor assay. Both ALDH1 positive and negative cell populations were isolated from the MG63 cell line, by cell-sorting using FACSAria. Both subpopulations had a comparable ability to differentiate similarly to the parental MG63, under normal monolayer culture condition. Subpopulations with the ability to form spheres in anchorage-independent, serum-starved conditions showed increased ALDH1 mRNA expression in addition to strong mRNA expression of the stem cell-related genes, such as Nanog, Oct3/4, Stat3 and Sox2, and possessed ability for self-renewal with secondary sphere formation. Sarcosphere cells from the MG63 cell line showed strong chemo-resistance against both doxorubicin and cisplatin compared with monolayer, adherent cells. In conclusion, sphere-forming cells with elevated ALDH1 are a possible candidate for sarcoma stem cells, possessing strong chemo-resistant capacities. Although ALDH1 elevation was not sufficient for representing sarcoma stem cells, the efficient detoxification could contribute to the chemo-resistant properties of the stem-like sphere cells form human sarcoma.
  • Masaki Tajiri; Naoko Wakabayashi; Megumu Tsujino; Minako Fujii; Kyoko Okabe; Kanya Honoki; Toshifumi Tsujiuchi
    Pathobiology 77 4 225 - 229 2010年07月 [査読有り]
     
    Objective: Germ line mutations of the LKB1 gene cause the autosomal dominant Peutz-Jeghers syndrome (PJS), and PJS has also been associated with an increased risk of developing cancers, suggesting LKB1 may act as a tumor suppressor in PJS. By contrast, LKB1 mutations are rare events in most sporadic tumors in non-PJS patients, except for lung cancers. To better understand the involvement of LKB1 gene alterations during lung carcinogenesis, we investigated the LKB1 gene mutations and expressions in lung adenocarcinomas induced by N-nitrosobis(2-hydroxypropyl)amine (BHP) in rats. Methods: Male Wistar rats, 6 weeks old, were given 2,000 ppm BHP in their drinking water for 12 weeks and maintained without further treatment until they were sacrificed at 25 weeks. A total of 15 lung adenocarcinomas were obtained, and genomic DNA was extracted for the search of mutations using polymerase chain reaction (PCR)-single strand conformation polymorphism analysis. To assess altered expressions of the LKB1 gene, reverse transcription-PCR analysis was also performed. Results: No mutations were found throughout exons 1-9 in any of the tumors. Aberrant transcripts bearing deletions of nucleotides 216-1459, 289-1302, 268-1261, or 257-1378 were detected in 5 of 15 adenocarcinomas (33.3%). Conclusion: These results suggest that alterations of the LKB1 gene might be involved in the development of lung adenocarcinomas induced by BHP in rats. Copyright © 2010 S. Karger AG.
  • Naoko Wakabayashi; Megumu Tsujino; Masaki Tajiri; Midori Taki; Ayumi Koshino; Hiroko Ikeda; Nobuyuki Fukushima; Toshifumi Tsujiuchi
    Journal of Toxicologic Pathology 23 1 63 - 66 2010年04月 [査読有り]
     
    Lysophosphatidic acid (LPA) is a bioactive phospholipid that stimulates cell proliferation and migration, and protects cells from apoptosis. It interacts with specific G protein-coupled transmembrane receptors. Recently, frequent mutations of the LPA receptor-1 (LPA1) gene were detected in rat lung adenocarcinomas induced by N-nitrosobis(2-hydroxypropyl)amine (BHP). In this study, to evaluate the involvement of other LPA receptor gene alterations during lung carcinogenesis, we investigated mutations of the LPA2, LPA3, LPA4 and LPA5 genes in lung adenocarcinomas induced by BHP in rats. Fifteen male Wistar rats, 6 weeks of age, were given 2000 ppm BHP in their drinking water for 12 weeks and then maintained without further treatment until sacrifice at 25 weeks, and 15 adenocarcinomas were obtained. Genomic DNAs were extracted from frozen tissues, and the LPA2, LPA3, LPA4 and LPA5 genes were examined for mutations, using polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP) analysis. No mutations of LPA2, LPA3, LPA4 and LPA5 were detected in the 15 adenocarcinomas. These results suggest that alterations due to LPA2, LPA3, LPA4 and LPA5 gene mutations might not be involved in the development of lung adenocarcinomas induced by BHP in rats.
  • Kyoko Okabe; Mai Hayashi; Minako Fujii; Kanya Honoki; Toshio Mori; Nobuyuki Fukushima; Toshifumi Tsujiuchi
    PATHOBIOLOGY 77 5 278 - 282 2010年 [査読有り]
     
    Objective: Lysophosphatidic acid (LPA), which is a bioactive phospholipid, interacts with specific G protein-coupled transmembrane receptors. Recently, alterations in LPA receptor genes have been reported in some tumor cells. In this study, to assess an involvement of LPA receptor genes in the development of human cancer cells, we looked for the presence of mutations in LPA receptor 1-6 (LPA1-6) genes in MG63 osteosarcoma, HT1080 fibrosarcoma, A549 lung adenocarcinoma, MCF-7 breast carcinoma, and G-361 melanoma cells. Methods: Genomic DNAs were extracted from each cell and polymerase chain reaction-single-strand conformation polymorphism analysis was performed to identify the mutations. Results: MG63 showed mutations in LPA1 and LPA3 genes, while no mutations in the LPA receptor genes were found in HT1080, A549, MCF-7 and G-361 cells. Sequence analysis revealed a CGC to CGT (Arg to Arg) transition at codon 314 in LPA1, and a GCG to GTG (Ala to Val) transition at codon 247 in LPA3. Conclusion: These results indicated that the mutations in LPA1 and LPA3 genes occur in MG63 cells, suggesting that the alterations in LPA receptor genes may play some role in the pathogenesis in human osteosarcoma cells. Copyright (C) 2010 S. Karger AG, Basel
  • Kyoko Okabe; Mai Hayashi; Naoko Wakabayashi; Yasuna Yamawaki; Miki Teranishi; Nobuyuki Fukushima; Toshifumi Tsujiuchi
    PATHOBIOLOGY 77 6 309 - 314 2010年 [査読有り]
     
    Objective: Lysophosphatidic acid (LPA) receptors act as several biological effectors through LPA, which is a bioactive phospholipid. Recently, aberrant expressions of LPA receptor genes due to DNA methylation have been detected in several tumor cells. In this study, we measured expression levels and DNA methylation status of LPA receptor genes in mouse tumor cells, LL/2 lung carcinoma, B16F0 melanoma, FM3A mammary carcinoma and L1210 leukemia cells, compared with normal tissues. Methods: Total RNAs were extracted and RT-PCR analysis was performed. For DNA methylation status, bisulfite sequencing analysis was carried out, comparing outcomes with other tumor cells and normal tissues. Results: The expressions of LPA1 gene were shown in LL/2, but not in B16F0, FM3A and L1210 cells. While the LPA2 gene was expressed in all 4 tumor cells, the LPA3 gene was unexpressed in them. The LPA1 and LPA3 unexpressed cells were highly methylated, although normal tissues were all unmethylated. The DNA methylation status was correlated with gene expression levels in cancer cells. Conclusion: The present results demonstrate that DNA methylation patterns of LPA receptor genes are dependent on cancer cell types, suggesting that LPA receptors may be new molecular targets for therapeutic approaches and chemoprevention. Copyright (C) 2011 S. Karger AG, Basel
  • Mami Furukawa; Ayako Yamasaki; Junichi Yoshida; Megumu Tsujino; Naoko Wakabayashi; Kanya Honoki; Toshifumi Tsujiuchi
    ANTICANCER RESEARCH 29 10 4047 - 4050 2009年10月 [査読有り]
     
    To evaluate an involvement of LKB1 gene alteration during pancreatic carcinogenesis, mutations in the LKB1 gene in hamster pancreatic duct adenocarcinomas (PDAs) induced by N-nitrosobis(2-oxopropyl)amine (BOP) were investigated. Female Syrian golden hamsters received 30 mg/kg of BOP followed by repeated exposure to an augmentation pressure regimen consisting of a choline-deficient diet combined with DL-ethionine then L-methionine and a further administration of 20 mg/kg BOP. A total of 10 PDAs obtained 10 weeks after beginning the experiment were examined for mutations using reverse transcription (RT)-polymerase chain reaction (PCR)-single-strand conformation polymorphism (SSCP) analysis. Mutations were detected in 3 out of the 10 PDAs (30.0%). Sequence analysis revealed the identity of these mutations to be a CCC to CCT (Pro to Pro) transition at codon 221, a CCG to CAG (Pro to Gln) transversion at codon 324 and a GAC to GGC (Asp to Gly) transition at codon 343. The LKB1 gene may be involved in the development of PDAs induced by BOP in hamsters.
  • S. Nishimura; O. Fukushima; H. Ishikura; T. Takahashi; M. Matsunami; T. Tsujiuchi; F. Sekiguchi; M. Naruse; Y. Kamanaka; A. Kawabata
    GUT 58 6 762 - 770 2009年06月 [査読有り]
     
    Objective: Hydrogen sulfide (H(2)S) is formed from L-cysteine by multiple enzymes including cystathionine-gamma-lyase (CSE) in mammals, and plays various roles in health and disease. Recently, a pronociceptive role for H(2)S in the processing of somatic pain was identified. Here, the involvement of H(2)S in pancreatic pain is examined. Methods: Anaesthetised rats or mice received an injection of NaHS, a donor for H(2)S, or capsaicin into the pancreatic duct, and the expression of spinal Fos protein was detected by immunohistochemistry. Pancreatitis was created by 6 hourly doses of caerulein in unanaesthetised mice, and pancreatitis-related allodynia/hyperalgesia was evaluated using von Frey hairs. CSE activity and protein levels in pancreatic tissues were measured using the colorimetric method and western blotting, respectively. Results: Either NaHS or capsaicin induced the expression of Fos protein in the superficial layers of the T8 and T9 spinal dorsal horn of rats or mice. The induction of Fos by NaHS but not capsaicin was abolished by mibefradil, a T-type Ca(2+) channel blocker. In conscious mice, repeated doses of caerulein produced pancreatitis accompanied by abdominal allodynia/hyperalgesia. Pretreatment with an inhibitor of CSE prevented the allodynia/hyperalgesia, but not the pancreatitis. A single dose of mibefradil reversed the established pancreatitis-related allodynia/hyperalgesia. Either the activity or protein expression of pancreatic CSE increased after the development of caerulein-induced pancreatitis in mice. Conclusions: The data suggest that pancreatic NaHS/H(2)S most probably targets T-type Ca(2+) channels, leading to nociception, and that endogenous H(2)S produced by CSE and possibly T-type Ca(2+) channels are involved in pancreatitis-related pain.
  • Nobuyuki Fukushima; Daisuke Furuta; Yuji Hidaka; Ryutaro Moriyama; Toshifumi Tsujiuchi
    JOURNAL OF NEUROCHEMISTRY 109 3 683 - 693 2009年05月 [査読有り]
     
    Many studies have shown that microtubules (MTs) interact with MT-associated proteins and motor proteins. These interactions are essential for the formation and maintenance of the polarized morphology of neurons and have been proposed to be regulated in part by highly diverse, unusual post-translational modifications (PTMs) of tubulin, including acetylation, tyrosination, detyrosination, Delta 2 modification, polyglutamylation, polyglycylation, palmitoylation, and phosphorylation. However, the precise mechanisms of PTM generation and the properties of modified MTs have been poorly understood until recently. Recent PTM research has uncovered the enzymes mediating tubulin PTMs and provided new insights into the regulation of MT-based functions. The identification of tubulin deacetylase and discovery of its specific inhibitors have paved the way to understand the roles of acetylated MTs in kinesin-mediated axonal transport and neurodegenerative diseases such as Huntington's disease. Studies with tubulin tyrosine ligase (TTL)-null mice have shown that tyrosinated MTs are essential in normal brain development. The discovery of TTL-like genes encoding polyglutamylase has led to the finding that polyglutamylated MTs which accumulate during brain development are involved in synapse vesicle transport or neurite outgrowth through interactions with motor proteins or MT-associated proteins, respectively. Here we review current exciting topics that are expected to advance MT research in the nervous system.
  • Hiromasa Fujii; Kanya Honoki; Toshifumi Tsujiuchi; Akira Kido; Kazuhiro Yoshitani; Yoshinori Takakura
    INTERNATIONAL JOURNAL OF ONCOLOGY 34 5 1381 - 1386 2009年05月 [査読有り]
     
    The presence of cancer stem cells in both solid and hematopoietic malignancies, has been recently linked to their pathogenesis. Sarcomas are rare, and diversely characterized by degrees of mesenchymal differentiation. The aim of the current study was to demonstrate whether the human sarcoma cell lines, osteosarcoma MG63, Ewing's sarcoma HTB166, fibrosarcoma HT1080, possess the stem-like properties which may contribute to the drug-resistance. All cell lines possessed an ability to form spherical, clonal expanding colonies (sarcospheres) in anchorage-independent, serum-starved conditions. Sarcospheres showed the stem-like properties with the ability of self-renewal, and increased expression of the stem cell-related genes such as Nanog, OCT3/4 SOX2 and DNA repair enzyme genes, MLH1 and MSH2. Sarcospheres showed strong resistance to doxorubicin and cisplatin, and caffeine, a DNA repair inhibitor, enhanced the efficacy of those drugs, suggesting that the drug resistance in sarcosphere cells was partly related to the efficient DNA repair ability. These results indicate that human sarcoma cell lines contain stem-like cell populations with strong drug resistance, and DNA repair inhibitor could enhance the efficacy of chemo-drugs against sarcomas.
  • Nobuyuki Fukushima; Daisuke Furuta; Yuji Hidaka; Ryutaro Moriyama; Toshifumi Tsujiuchi
    JOURNAL OF NEUROCHEMISTRY 109 3 683 - 693 2009年05月 
    Many studies have shown that microtubules (MTs) interact with MT-associated proteins and motor proteins. These interactions are essential for the formation and maintenance of the polarized morphology of neurons and have been proposed to be regulated in part by highly diverse, unusual post-translational modifications (PTMs) of tubulin, including acetylation, tyrosination, detyrosination, Delta 2 modification, polyglutamylation, polyglycylation, palmitoylation, and phosphorylation. However, the precise mechanisms of PTM generation and the properties of modified MTs have been poorly understood until recently. Recent PTM research has uncovered the enzymes mediating tubulin PTMs and provided new insights into the regulation of MT-based functions. The identification of tubulin deacetylase and discovery of its specific inhibitors have paved the way to understand the roles of acetylated MTs in kinesin-mediated axonal transport and neurodegenerative diseases such as Huntington's disease. Studies with tubulin tyrosine ligase (TTL)-null mice have shown that tyrosinated MTs are essential in normal brain development. The discovery of TTL-like genes encoding polyglutamylase has led to the finding that polyglutamylated MTs which accumulate during brain development are involved in synapse vesicle transport or neurite outgrowth through interactions with motor proteins or MT-associated proteins, respectively. Here we review current exciting topics that are expected to advance MT research in the nervous system.
  • Yumi Obo; Takanori Yamada; Mami Furukawa; Mayuko Hotta; Kanya Honoki; Nobuyuki Fukushima; Toshifumi Tsujiuchi
    MUTATION RESEARCH-FUNDAMENTAL AND MOLECULAR MECHANISMS OF MUTAGENESIS 660 1-2 47 - 50 2009年01月 [査読有り]
     
    Lysophosphatidic acid (LPA) is a bioactive phospholipid that stimulates cell proliferation, migration, and protects cells from apoptosis. It interacts with Specific G protein-coupled transmembrane receptors, including LPA1 to LPA5. In the present study, to clarify an involvement of LPA I gene alterations in the development of hepatocellular carcinomas (HCCs) we investigated the LPA1 mutations in rat HCCs induced by exogenous and endogenous liver carcinogenesis models. We induced HCCs in rats with N-nitrosodiethylamine (DEN) and a choline-deficient L-amino acid-defined (CDAA) diet. RNAs were extracted from 15 HCCS induced by DEN and 12 HCCs induced by the CDAA diet. To identify LPA1 mutations, reverse transcription (RT) - polymerase chain reaction (PCR) - single strand conformation polymorphism (SSCP) analysis, followed by nucleotide sequencing, was performed. Missense mutations were detected in 7 out of 15 HCCs (46.7%,) induced by DEN. Five Out of 12 HCCs (41.7%,) induced by the CDAA diet also showed missense mutations. These results demonstrated that mutations in LPA1 gene occur in rat HCCs induced by DEN and the CDAA diet, suggesting that LPA1 mutations may be essentially involved ill rat liver carcinogenesis. (c) 2008 Elsevier B.V. All rights reserved.
  • Takanori Yamada; Yumi Obo; Mami Furukawa; Mayuko Hotta; Ayako Yamasaki; Kanya Honoki; Nobuyuki Fukushima; Toshifumi Tsujiuchi
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 378 3 424 - 427 2009年01月 [査読有り]
     
    Lysophosphatidic acid (LPA) is a bioactive phospholipid that stimulates cell proliferation, migration, and protects cells from apoptosis. It interacts with specific G protein-coupled transmembrane receptors. In this study, mutations of lysophosphatidic acid receptor-1 (LPA1) gene were investigated to clarify the possible molecular mechanisms underlying the development of lung tumors induced by N-nitrosobis(2-hydroxypropyl)amine (BHP) in rats. Male Wistar rats, 6 weeks of age. were given 2000 ppm BHP in their drinking water for 12 weeks and then maintained without further treatment until sacrifice at 25 weeks. Genomic DNAs were extracted from paraffin-embedded tissues and exons 2-4 were examined for mutations, using polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP) analysis. No LPA1 mutations were detected in 15 hyperplasias, but 2 out of 12 adenomas (16.7%) and 7 Out Of 17 adenocarcinomas (41.2%). These results suggest that mutations of LPA1 gene may be involved in the acquisition of growth advantage from adenomas to adenocarcinomas in lung carcinogenesis induced in rats by BHP. (C) 2008 Elsevier Inc. All rights reserved.
  • Toshifumi Tsujiuchi; Mami Furukawa; Yumi Obo; Ayako Yamasaki; Mayuko Hotta; Chie Kusunoki; Naoko Suyama; Toshio Mori; Kanya Honoki; Nobuyuki Fukushima
    JOURNAL OF TOXICOLOGIC PATHOLOGY 22 1 89 - 92 2009年 [査読有り]
     
    To evaluate the involvement of lysophosphatidic acid receptor-1 (LPA1) gene alteration in pancreatic carcinogenesis, we investigated mutations in the LPA1 gene in hamster pancreatic duct adenocarcinomas (PDAs) and established cell lines. Female Syrian golden hamsters received 30 mg/kg of N-nitrosobis(2-oxopropyl) amine (BOP) followed by repeated exposure to an augmentation pressure regimen consisting of a choline-deficient diet combined with DL-ethionine and then L-methionine and a further administration of 20 mg/kg BOP. A total of 10 PDAs obtained 10 weeks after beginning the experiment and three cell lines established from subcutaneously transplantable PDAs in syngeneic hamsters were examined for mutations using reverse transcription-polymerase chain reaction-single strand conformation polymorphism (RT-PCR-SSCP) analysis. A mutation was detected in only one PDA (1/10, 10%) in the form of a GGA to GTA (Gly to Val) transversion at codon 355, and no mutations were detected in the three cell lines. These results suggest that the LPA1 gene mutation may play roles in a limited fraction of BOP-induced pancreatic duct carcinogenesis in hamsters. (J Toxicol Pathol 2009; 22: 89-92)
  • Shinya Shano; Kazuki Hatanaka; Shinsuke Ninose; Ryutaro Moriyama; Toshifumi Tsujiuchi; Nobuyuki Fukushima
    BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH 1783 5 748 - 759 2008年05月 [査読有り]
     
    Lysophosphatidic acid (LPA) is an extracellular signaling lipid that regulates cell proliferation, survival, and motility of normal and cancer cells. These effects are produced through G protein-coupled LPA receptors, LPA(1) to LPA(5). We generated an LPA(1) mutant lacking the SerValVal sequence of the C-terminal PDZ-binding domain to examine the role of this domain in intracellular signaling and other cellular functions. B103 neuroblastoma cells expressing the mutant LPA(1) showed rapid cell proliferation and tended to form colonies under serum-free conditions. The enhanced cell proliferation of the mutant cells was inhibited by exogenous expression of the plasmids inhibiting G proteins including G(beta gamma), G(alpha i) and G(alpha q) or G(alpha 12/13), or treatment with pertussis toxin, phosphoinositide 3-kinase (PI3K) inhibitors or a Rho inhibitor. We confirmed that the PI3K-Akt and Rho pathways were intrinsically activated in mutant cells by detecting increases in phosphorylated Akt in western blot analyses or by directly measuring Rho activity. Interestingly, expression of the mutant LPA(1) in non-tumor mouse fibroblasts induced colony formation in a clonogenic soft agar assay, indicating that oncogenic pathways were activated. Taken together, these observations suggest that the mutant LPA(1) constitutively activates the G protein signaling leading to PI3K-Akt and Rho pathways, resulting in enhanced cell proliferation. (C) 2007 Elsevier B.V. All rights reserved.
  • Shinya Shano; Kazuki Hatanaka; Shinsuke Ninose; Ryutaro Moriyama; Toshifumi Tsujiuchi; Nobuyuki Fukushima
    Biochimica et biophysica acta 1783 5 748 - 59 2008年05月 
    Lysophosphatidic acid (LPA) is an extracellular signaling lipid that regulates cell proliferation, survival, and motility of normal and cancer cells. These effects are produced through G protein-coupled LPA receptors, LPA(1) to LPA(5). We generated an LPA(1) mutant lacking the SerValVal sequence of the C-terminal PDZ-binding domain to examine the role of this domain in intracellular signaling and other cellular functions. B103 neuroblastoma cells expressing the mutant LPA(1) showed rapid cell proliferation and tended to form colonies under serum-free conditions. The enhanced cell proliferation of the mutant cells was inhibited by exogenous expression of the plasmids inhibiting G proteins including G(betagamma), G(alphai) and G(alphaq) or G(alpha12/13), or treatment with pertussis toxin, phosphoinositide 3-kinase (PI3K) inhibitors or a Rho inhibitor. We confirmed that the PI3K-Akt and Rho pathways were intrinsically activated in mutant cells by detecting increases in phosphorylated Akt in western blot analyses or by directly measuring Rho activity. Interestingly, expression of the mutant LPA(1) in non-tumor mouse fibroblasts induced colony formation in a clonogenic soft agar assay, indicating that oncogenic pathways were activated. Taken together, these observations suggest that the mutant LPA(1) constitutively activates the G protein signaling leading to PI3K-Akt and Rho pathways, resulting in enhanced cell proliferation.
  • Kyoko Shimizu; Yumi Itsuzaki; Hiromasa Fujii; Kanya Honoki; Toshifumi Tsujiuchi
    MOLECULAR CARCINOGENESIS 47 2 80 - 87 2008年02月 [査読有り]
     
    Alterations of the Rassf1a gene were investigated in pancreatic duct adenocarcinomas (PDAs) induced by N-nitrosobis(2-oxopropyl)amine (BOP) in hamsters. Female Syrian golden hamsters received 70 mg/kg BOP, followed by repeated exposures to an augmentation pressure regimen consisting of a choline-deficient diet combined with a sequential course of DL-ethionine, L-methionine, and 20 mg/kg BOP. A total of 15 PDAs were obtained, and total RNAs were assessed by real-time quantitative reverse transcription (RT)-polymerase chain reaction (PCR). Expression of the Rassf1a was significantly reduced in PDAs (P<0.005) compared with normal pancreatic tissues. For analysis of methylation status, bisulfite sequencing was performed. Normal tissues were all unmethylated in the 5' upstream region of Rassf1a. In contrast, four PDAs were highly methylated, correlating with reduced expression of the Rassf1a gene. Using reverse transcription (RT)-polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP) analysis, mutations were detected in 3 out of 15 PDAs (20%). These results suggested that alterations of the Rassf1a gene may be involved in development of PDAs induced by BOP in hamsters. (C) 2007 Wiley-Liss, Inc.
  • Mariko Onishi; Mariko Saito; Yui Sokuza; Chiharu Mori; Tomoki Nishikawa; Kyoko Shimizu; Eriko Sugata; Toshifurni Tsujiuchi
    MUTATION RESEARCH-FUNDAMENTAL AND MOLECULAR MECHANISMS OF MUTAGENESIS 638 1-2 133 - 138 2008年02月 [査読有り]
     
    Mutations in the mitochondrial DNA (mtDNA) displacement loop (D-loop) region were investigated to clarify the possible molecular mechanisms underlying the development of lung tumors induced by N-nitrosobis(2-hydroxypropyl)amine (BHP) in rats. Male Wistar rats, 6 weeks of age, were given 2000 ppm BHP in their drinking water for 12 weeks and then maintained without further treatment until sacrifice at 25 weeks. Genomic DNA was extracted from paraffin-embedded tissues and polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP) analysis, followed by nucleotide sequencing, was performed. Eleven out of 24 hyperplasias (45.6%), 8 out of 16 adenomas (50.0%), and 14 out of 21 adenocarcinomas (66.7%) showed numerical changes, in a polymeric C-tract at positions 16,086-16,092 of the mtDNA D-loop, with a one base insertion of cytosine increasing the length of the C-tract, from the seven nucleotides observed in normal lung tissues from non-BHP treated rats, to eight. These changes were all homoplasmic and no changes were found in lung lesions when the length of the C-tract in the normal lung tissues adjacent to the lesions was seven. These results suggest that alterations in the mtDNA D-loop may occur in an early phase of lung carcinogenesis induced in rats by BHP. (C) 2007 Elsevier B.V. All rights reserved.
  • Mariko Onishi; Mariko Saito; Yui Sokuza; Chiharu Mori; Tomoki Nishikawa; Toshio Mori; Toshifumi Tsujiuchi
    Journal of Toxicologic Pathology 20 4 263 - 266 2008年01月 [査読有り]
     
    Mutations in the mitochondrial DNA (mtDNA) displacement loop (D-loop) region were investigated to clarify the possible molecular mechanisms underlying the development of hamster pancreatic tumors. Female Syrian golden hamsters received 30 mg/kg N-nitrosobis(2-oxopropyl)amine (BOP), followed by repeated exposure to an augmentation pressure regimen consisting of a choline-deficient diet combined with ethionine, then methionine injections and a further administration of 20 mg/kg BOP. A total of 11 pancreatic duct adenocarcinomas (PDAs) were obtained 10 weeks after beginning the experiment, and three cell lines established from subcutaneously transplantable PDAs in syngeneic hamsters were examined for mutations using polymerase chain reaction (PCR) - single strand conformation polymorphism (SSCP) analysis. No mutations were detected in the 11 PDAs and 3 cell lines. These results suggest that alterations of the mtDNA D-loop might not be involved in BOP-induced pancreatic duct carcinogenesis in hamsters.
  • Hiromasa Fuji; Kanya Honoki; Toshifumi Tsujiuch; Akira Kido; Kazuhiro Yoshitani; Yoshinori Takakura
    IN VIVO 22 1 21 - 25 2008年01月 [査読有り]
     
    2-Methoxyestradiol (2-ME) has been found to possess antitumor activity in vivo and in vitro. It has been suggested that 2-ME induces apoptosis resulting in G(2)/M arrest of tumor cells. In this study, the effect of 2-ME was evaluated in rat osteosarcoma and malignant fibrous histiocytoma (MFH) cell lines. 2-ME was used at final concentrations of 100 nM to 2 mu M. The effect of 2-ME on cell growth was measured by the MTS assay. Induction of apoptosis and activation of caspase-3 were investigated along with apoptosis-related gene expression. The data showed that 2-ME significantly inhibited cell growth, inducing apoptosis. The activity of caspase-3 was increased at 20 h and 40 h in both cell lines. 2-ME induced p16 expression, which was possibly involved in the apoptotic process. These results suggested that the 2-ME-induced apoptosis of rat osteosarcoma and rat MFH cells was accompanied by caspase-3 activation through p16 induction.
  • Yui Sokuza; Takanori Yamada; Yumi Obo; Mami Furukawa; Mayuko Hotta; Ayako Yamasaki; Kanya Honoki; Toshifumi Tsujiuchi
    JOURNAL OF TOXICOLOGIC PATHOLOGY 21 4 257 - 260 2008年 [査読有り]
     
    Protocadherins are a major subfamily of the cadherin superfamily that play an important role in communication between adjacent cells. To clarify the involvement of Protocadherin 20 (Pcdh20) gene in rat lung carcinogenesis, we investigated the expression of Pcdh20 and its methylation status in rat lung adenocarcinomas induced by N-nitrosobis(2-hydroxypropyl)amine (BHP). Six-week old male Wistar rats were given 2000 ppm BHP in their drinking water for 12 weeks and were maintained without further treatment until they were sacrificed after 25 weeks. Total RNAs were extracted from 7 lung adenocarcinomas, one from each BHP-treated rat, and the expression levels of Pcdh20 were measured using semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis. Four out of 7 tumors showed reduced expression of Pcdh20, compared with 3 normal lung tissues. For methylation analysis, bisulfite sequencing was performed. The 5' upstream region of Pcdh20 was methylated in 4 adenocarcinomas with reduced expression of Pcdh20, but was unmethylated in normal lung tissue. These results suggest that aberrant methylation of the Pcdh20 gene might be involved in the development of lung adenocarcinomas induced by BHP in rats. (J Toxicol Pathol 2008; 21: 257-260)
  • Toshifumi Tsujiuchi; Tomomi Masaoka; Eriko Sugata; Mariko Onishi; Hiromasa Fujii; Kyoko Shimizu; Kanya Honoki
    MOLECULAR CARCINOGENESIS 46 10 819 - 823 2007年10月 [査読有り]
     
    DAL-1 (differentially expressed in adenocarcinoma of the lung) is an actin-binding protein that has been shown to suppress growth in lung cancer cells. Recently, inactivation of the gene encoding DAL-1 due to hypermethylation has been found in several human malignancies, including lung cancers. To assess the involvement of the Dal-1 gene in rat lung carcinogenesis, we investigated the expression of Dall and its methylation status in rat lung adenocarcinomas induced by N-nitrosobis(2-hydroxypropyl)amine (BHP). Six-week old male Wistar rats (n = 11) were given 2000 ppm BHP in their drinking water for 12 wk and maintained without further treatment until they were sacrificed at 25 wk. Total RNA was extracted from 11 lung adenocarcinomas, one from each BHP treated rat, and Dal-1 gene expression was analyzed using real-time quantitative reverse transcription-polymerase chain reaction. Dal-1 expression was significantly reduced in the lung adenocarcinomas compared with three normal lung tissues (P < 0.05). For methylation analysis, bisulfite sequencing was performed using normal lung tissue and tissue from 4 tumors, all of which showed reduced expression of Dal-1. The 5' upstream region was highly methylated in all four adenocarcinomas, whereas this region was unmethylated in normal lung tissue. These results suggest that aberrant methylation of the Dal-1 gene might be involved in the development of lung adenocarcinomas induced in rats by BHP. (c) 2007 Wiley-Liss, Inc.
  • Mariko Onishi; Yui Sokuza; Tomoki Nishikawa; Chiharu Mori; Kimiko Uwataki; Kanva Honoki; Toshifumi Tsujiuchi
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 362 1 183 - 187 2007年10月 [査読有り]
     
    Mutations of the mitochondria DNA (mtDNA) displacement loop (D-loop) were investigated to clarify different changes of exogenous and endogenous liver carcinogenesis in rats. We induced hepatocellular carcinomas (HCCs) in rats with N-nitrosodiethylamine (DEN) and a choline-deficient L-amino acid-defined (CDAA) diet. DNAs were extracted from 10 HCCs induced by DEN and 10 HCCs induced by the CDAA diet. To identify mutations in mtDNA D-loop, polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP) analysis, followed by nucleotide sequencing, was performed. Mutations were detected in 5 out of 10 HCCs (50%) induced by DEN. Four out of 5 mutations were G/C to A/T transitions at positions 15707, 15717, 15930, and 16087, and one T/A to C/G transition at position 15559. By contrast, no mutations were found in 10 HCCs induced by the CDAA diet. These results demonstrated that mutations in mtDNA D-loop occur in rat HCCs induced by DEN but not by the CDAA diet, suggesting that mtDNA D-loop is a target of exogenous liver carcinogenesis in rats. (C) 2007 Elsevier Inc. All rights reserved.
  • Hiromasa Fujii; Kanya Honoki; Toshifumi Tsujiuchi; Akira Kido; Kazuhiro Yoshitani; Toshio Mori; Yoshinori Takakura
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 362 3 773 - 778 2007年10月 [査読有り]
     
    The presence of cancer stem cells, in both hematopoietic and solid malignancies, has been recently linked to their pathogenesis. We aimed to identify the characteristics and stem-like properties of sphere-colony forming cells in rat osteosarcoma and malignant fibrous histiocytoma cell lines. The results showed that both cell lines possessed an ability to form spherical, clonally expanding colonies in anchorage-independent, serum-starved conditions in N2/1% methyleellulose medium. The sphere cells showed stem-like properties with the ability to self-renew, and expressed the stem cell-related STAT3 and Bmil genes. Interestingly, spheres from both sarcomas remarkably decreased the expression of INK4a/ARF locus genes, p16(INK4a) and p19(ARF), which could be related to the resistance against cell senescence and apoptosis. Spheres showed strong tumorigenicity with metastatic potential in vivo via the inoculation into syngeneic rats, suggesting the presence of these populations might contribute to the tumor development such as metastasis via the resistance to apoptotic stimuli. (c) 2007 Elsevier Inc. All rights reserved.
  • Kyoko Shimizu; Mariko Onishi; Eriko Sugata; Yui Sokuza; Chiharu Mori; Tomoki Nishikawa; Kanya Honoki; Toshifumi Tsujiuchi
    CANCER SCIENCE 98 9 1318 - 1322 2007年09月 [査読有り]
     
    The authors investigated the DNA methylation patterns of the E-cadherin, Connexin 26 (Cx26), Rassf1a and c-fos genes in the early phase of rat hepatocarcinogenesis induced by a choline-deficient L-amino acid-defined (CDAA) diet. Six-week-old F344 male rats were continuously fed with the CDAA diet, and three animals were then killed at each of 4 and 8 days and 3 weeks. Genomic DNA was extracted from livers for assessment of methylation status in the 5' upstream regions of E-cadherin, Cx26, Rassf1a and c-fos genes by bisulfite sequencing, compared with normal livers. The livers of rats fed the CDAA diet for 4 and 8 days and 3 weeks were methylated in E-cadherin, Cx26 and Rassf1a genes, while normal livers were all unmethylated. In contrast, normal livers were highly methylated in c-fos gene. Although the livers at 4 days were weakly methylated, those at 8 days and 3 weeks were markedly unmethylated. Methylation patterns of CpG sites in E-cadherin, Cx26 and Rassf1a were sparse and the methylation was not associated with gene repression. These results indicate that gene-specific DNA methylation patterns were found in livers of rats after short-term feeding of the CDAA diet, suggesting gene-specific hypermethylation might be involved in the early phase of rat hepatocarcinogenesis induced by the CDAA diet.
  • Kanya Honoki; Elizabeth Stojanovski; Mark McEvoy; Hiromasa Fujii; Toshifumi Tsujiuchi; Akira Kido; Yoshinori Takakura; John Attia
    CANCER 110 6 1351 - 1360 2007年09月 [査読有り]
     
    BACKGROUND. Despite findings from individual studies regarding prognostic factors for Ewing sarcoma, no conclusive results have been produced, partly because of small sample sizes. The objective of the current study was to evaluate whether the presence of p16(INK4a) alteration is associated with a poorer prognosis in patients with Ewing sarcomas. METHODS. A review was conducted of publications that assessed associations between P16(INK4a) a status and 2-year survival among patients with Ewing sarcoma. The association between metastatic disease at initial diagnosis and 2-year survival was evaluated by synthesizing data in the form of risk ratios. RESULTS. Of 11 studies that were identified in the initial search strategy, 6 studies, representing 188 patients, met the inclusion criteria and, consequently, were pooled for quantitative analyses. The estimated pooled risk ratio of p16(INK4a) aberration was 2.17 (95% confidence interval [95% CI], 1.55-3.03; P < .001), whereas the estimated pooled risk ratio of metastasis at diagnosis among the 164 eligible patients was 2.60 (95% CI, 1.71-3.97; P < .001). There was no statistically significant difference in the pooled estimated risk ratios of p16(INK4a) aberration for a poor prognosis between patients with and without metastasis at diagnosis (1.86 and 2.21, respectively; P > .59). CONCLUSIONS. The presence of p16(INK4a) alteration was a statistically significant predictor of prognosis for patients with Ewing sarcoma. Along with other prognostic factors, such as metastasis, the p16(INK4a) alteration may be a potential candidate for improving the risk-stratifying strategy for patients with these tumors.
  • Toshifumi Tsujiuchi; Eriko Sugata; Tomomi Masaoka; Mariko Onishi; Hiromasa Fujii; Kyoko Shimizu; Kanya Honoki
    CANCER SCIENCE 98 7 943 - 948 2007年07月 [査読有り]
     
    To assess the involvement of the TSLC cascade in hepatocarcinogenesis, we investigated the expression and DNA methylation patterns of the genes Tslc1 and Dal-1 in hepatocellular carcinomas (HCC) induced using N-nitrosodiethylamine (DEN) in rats. Six-week-old male F344 rats received a single intraperitoneal injection of DEN at a dose of 10 mg/kg body weight, followed by combined treatment with partial hepatectomy and colchicine to induce cell-cycle disturbance and a selection procedure consisting of 2-acetylaminofluorene and carbon tetrachloride. Total RNA was extracted from 10 HCC, and the expression levels of Tslc1 and Dal-1 were measured using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis. Three of 10 HCC showed reduced expression of Tslc1, compared with normal liver tissues, but no changes in the expression level of Dal-1 were found. For DNA methylation analysis, bisulfite sequencing was performed. The 5' upstream region of Tslc1 was methylated in the three HCC in which its expression was reduced, but was unmethylated in normal liver tissue. Western blot analysis also revealed reduced expression of Tslc1 protein in the three HCC. These results suggest that alterations to the TSLC cascade might have a role in hepatocarcinogenesis using DEN in rats.
  • Hiroyasu Ishikura; Sachiyo Nishimura; Maho Matsunami; Toshifumi Tsujiuchi; Tsuyoshi Ishiki; Fumiko Sekiguchi; Mitsuhide Naruse; Toshio Nakatani; Yoshihisa Kamanaka; Atsufumi Kawabata
    LIFE SCIENCES 80 21 1999 - 2004 2007年05月 [査読有り]
     
    Camostat mesilate, an orally available proteinase inhibitor, is clinically used for treatment of pancreatitis. Given recent evidence that pancreatic proteinases including trypsin and/or proteinase-activated receptor-2 (PAR2) might be involved in pancreatic pain, we examined if camostat mesilate could suppress spinal Fos expression, a marker for neuronal activation, following specific application of trypsin to the pancreas, and pancreatitis-related referred allodynia. Trypsin, administered into the pancreatic duct, caused delayed expression of Fos proteins in the superficial layer of the bilateral T8 and T9 spinal dorsal horns in rats. The trypsin-induced spinal Fos expression was completely abolished by oral preadministration of camostat mesilate at 300 mg/kg. After hourly repeated (6 times in total) administration of caerulein, mice showed typical symptoms of pancreatitis, accompanied by mechanical allodynia in the upper abdomen (i.e., referred hyperalgesia/allodynia), as assessed by use of von Frey filaments. Camostat mesilate at 100-300 mg/kg, given orally twice before the 1st and 4th doses of cacrulein, abolished the pancreatitis-related abdominal allodynia, while it partially prevented the inflammatory signs. The same doses of camostat mesilate, when administered once after the final dose of caerulein, also revealed significant anti-allodynic effect. These data suggest that camostat mesilate prevents and/or depresses pancreatitis-induced pain and/or referred hyperalgesia/allodynia, in which proteinases including trypsin would play a critical role. (C) 2007 Elsevier Inc. All rights reserved.
  • Toshifumi Tsujiuchi; Kyoko Shimizu; Yumi Itsuzaki; Mariko Onishi; Eriko Sugata; Hiromasa Fujii; Kanya Honoki
    MOLECULAR CARCINOGENESIS 46 4 269 - 274 2007年04月 [査読有り]
     
    We investigated DNA methylation patterns of E-cadherin and Connexin26 (Cx26) genes in rat hepatocellular carcinomas (HCCs) induced by a choline-deficient L-Amino Acid-defined (CDAA) diet. Six-wks-old F344 male rats were continuously fed with a CDAA diet for 75 Wks, and were then killed. A total of five HCCs were obtained, and genomic DNA was extracted from each HCC for assessment of methylation status in the 5' upstream regions of E-cadherin and Cx26 genes by bisulfite sequencing, comparing to two normal liver tissues. The five HCCs showed highly methylated E-cadherin and Cx26 genes, while these genes in two normal liver tissues were all unmethylated. For analysis of gene expression, real-time quantitative reverse transcription (RT)-polymerase chain reaction (PCR) was performed. Expressions of E-cadherin and Cx26 genes were significantly reduced in the five HCCs (P < 0.0001 and P < 0.001, respectively) compared to normal liver tissues, correlating with their methylation statuses. These results suggested that hype rmethylati on of E-cadherin and Cx26 genes may be involved in the development of HCCs induced by a CDAA diet in rats. (c) 2007 Wiley-Liss, Inc.
  • Mariko Onishi; Kyoko Shimizu; Eriko Sugata; Mayumi Fujii; Midori Yoshida; Kanya Honoki; Toshifumi Tsujiuchi
    Journal of Toxicologic Pathology 20 1 65 - 69 2007年03月 [査読有り]
     
    Epidermal growth factor receptor (EGFR), a receptor protein tyrosine kinase, is a transmembrane protein. Recent studies indicate that mutations in the gene encoding EGFR are present in several human cancers. To assess the involvement of these mutations in the development of rat tumors, we looked for the presence of mutations in exons 18-21, a region which encodes the tyrosine kinase domain of Egfr, in lung and liver tumors in rats. Lung adenocarcinomas were induced in rats by exposure to N-nitrosobis(2-hydroxypropyl)amine (BHP). We also induced hepatocellular carcinomas (HCCs) in rats with multiple hepatocarcinogens and a choline-deficient L-amino acid-defined (CDAA) diet. Genomic DNA was extracted from 12 lung adenocarcinomas, 8 HCCs induced by multiple hepatocarcinogens and 8 HCCs induced by the CDAA diet. To identify mutations in Egfr, polymerase chain reaction (PCR)- single-strand conformation polymorphism (SSCP) analysis was performed. No mutations were detected throughout exons 18-21, in either lung or liver tumors in rats. These results suggest that alterations to Egfr might not be involved in the development of lung and liver tumors in rats.
  • Kyoko Shimizu; Mariko Onishi; Eriko Sugata; Hiromasa Fujii; Kanya Honoki; Toshifumi Tsujiuchi
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 353 2 522 - 526 2007年02月 [査読有り]
     
    To determine if the Tslc1 gene is involved in pancreatic carcinogenesis, the expression level of Tslc1 and the DNA methylation status of its 5' upstream region were investigated in pancreatic duct adenocarcinomas (PDAs) induced in hamsters by N-nitrosobis(2-oxopropyl)amine (BOP). Female Syrian golden hamsters received 70 mg/kg of BOP followed by repeated exposure to an augmentation pressure regimen consisting of a choline-deficient diet combined with ethionine-methionione-BOP injection. Total RNA was extracted from I I PDAs and the level of Tslc1 expression was measured in each by real-time quantitative reverse transcription (RT)-polymerase chain reaction (PCR). The expression level of Tslc1 was significantly reduced in PDAs (p < 0.05) compared with normal pancreatic tissues. In order to assess the DNA methylation status of the 5' upstream region of Tslc1, bisulfite sequencing was performed. Although this region was unmethylated in normal pancreatic tissue, it was highly methylated in four PDAs, correlating with reduced Tslc1 expression. These results suggest that a reduction in the expression of Tslc1 due to aberrant DNA methylation might be involved in the development of PDAs induced in hamsters by BOP. (c) 2006 Elsevier Inc. All rights reserved.
  • Toshifumi Tsujiuchi; Kyoto Shimizu; Mariko Onishi; Mayumi Shigemura; Shinya Shano; Kanya Honoki; Nobuyuki Fukushima
    Journal of Toxicologic Pathology 19 3 137 - 141 2006年10月 [査読有り]
     
    Lysophosphatidic acid (LPA) is a bioactive phospholipid that stimulates cell proliferation and migration, and protects cells from apoptosis. It interacts with at least three specific G protein-coupled transmembrane receptors, LPA1, LPA2 and LPA3. In the present study, the involvement of LPA receptor genes in the development of rat tumors was evaluated by measuring mRNA expression of the LPA1, LPA2 and LPA3 genes in nitroso-compound induced lung adenocarcinomas and hepatocellular carcinomas(HCCs) in rats. Total RNA was extracted from 12 lung adenocarcinomas and 6 HCCs, and expression analysis was performed by real-time quantitative reverse transcription (RT)-polymerase chain reaction (PCR). In lung adenocarcinomas, the expressions of LPA2 and LPA3 were significantly increased (p < 0.001 and p < 0.005, respectively) compared with normal lung tissue. In contrast, LPA1 showed a lower level of expression, but the difference was not significant. In HCCs, the expression of LPA2 was significantly increased (p < 0.05) compared with normal liver tissue. In contrast, varying levels of LPA1 and LPA3 expression were detected. The ratio of LPA2/LPA1, which is important for understanding the effects of LPA on target cells than the levels of LPA1 and LPA2 separately, increased markedly in both lung (p< 0.005) and liver tumors (p< 0.005). These results suggest that aberrant expressions of LPA receptors may be involved in the development of lung and liver tumors in the rat. © 2006 The Japanese Society of Toxicologic Pathology.
  • Toshifumi Tsujiuchi; Kyoko Shimizu; Mariko Onishi; Eriko Sugata; Hiromasa Fujii; Toshio Mori; Kanya Honoki; Nobuyuki Fukushima
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 349 3 1151 - 1155 2006年10月 [査読有り]
     
    Lysophosphatidic acid (LPA) is a bioactive phospholipid that stimulates cell proliferation, migration, and protects cells from apoptosis. It interacts with specific G protein-coupled transmembrane receptors. Recently, it has been reported that alterations of LPA receptor expression might be important in the malignant transformation of tumor cells. Therefore, to assess an involvement of DNA methylation in reduced expression of the LPA receptor-1 (lpa1) gene, we investigated the expression of the lpa1 gene and its DNA methylation patterns in rat tumor cell lines. Both rat brain-derived neuroblastoma B103 and liver-derived hepatoma RH7777 cells used in this study indicated no expression of lpa1. For the analysis of methylation status, bisulfite sequencing was performed with B103 and RH7777 cells, comparing with other lpa1 expressed cells and normal tissues of brain and liver. The lpa1 expressed cells and tissues were all unmethylated in this region of lpa1. In contrast, both B103 and RH7777 cells were highly methylated, correlating with reduced expression of the lpa1. Treatment with 5-aza 2'-deoxycytidine induced expression of lpa1 gene in B103 and RH7777 cells after 24 h. In RH7777 cells treated with 5-aza 2'-deoxycytidine, stress fiber formation was also observed in response to LPA in RH7777 cells, but not in untreated RH7777 cells. These results suggest that aberrant DNA methylation of the lpa1 gene may be involved in its reduced expression in rat tumor cells. (c) 2006 Elsevier Inc. All rights reserved.
  • Kyoko Shimizu; Yozo Shimoichi; Daisei Hinotsume; Yumi Itsuzaki; Hiromasa Fujii; Kanya Honoki; Toshifumi Tsujiuchi
    MOLECULAR CARCINOGENESIS 45 9 710 - 714 2006年09月 [査読有り]
     
    Gap junctions are mediated by intercellular channels that connect adjacent cells and are composed of Connexin (Cx) proteins. A member of the Cx family, Cx26 is considered a potential tumor suppressor in several cancers. The expression of Cx26 gene and its methylation status in rat lung adenocarcinomas induced by N-nitrosobis(2-hydroxypropyl)amine (BHP) were investigated. Six-wk-old male Wistar rats were given 2,000 ppm BHP in their drinking water for 12 wk and maintained without further treatment until they were sacrificed at 25 wk. A total of nine lung adenocarcinomas were obtained and total RNA was extracted to assess expression by real-time quantitative reverse transcription (RT)-polymerase chain reaction (PCR). Five out of nine adenocarcinomas showed reduced expression compared with normal lung tissue. We next performed a bisulfite sequence analysis to measure the methylation status of the 5 ' upstream region of the Cx26 gene in two normal lung tissues and five lung adenocarcinomas that showed reduced expression of Cx26. All five adenocarcinomas were highly methylated in the 5 ' upstream region, while the two normal lung tissues were unmethylated. This suggests that aberrant methylation of the Cx26 gene may be involved in the development of lung adenocarcinomas induced by BHP in rats. (c) 2006 Wiley-Liss, Inc.
  • Kyoko Shimizu; Yozo Shimoichi; Daisei Hinotsume; Yumi Itsuzaki; Hiromasa Fujii; Kanya Honoki; Toshifumi Tsujiuchi
    MOLECULAR CARCINOGENESIS 45 9 710 - 714 2006年09月 [査読有り]
     
    Gap junctions are mediated by intercellular channels that connect adjacent cells and are composed of Connexin (Cx) proteins. A member of the Cx family, Cx26 is considered a potential tumor suppressor in several cancers. The expression of Cx26 gene and its methylation status in rat lung adenocarcinomas induced by N-nitrosobis(2-hydroxypropyl)amine (BHP) were investigated. Six-wk-old male Wistar rats were given 2,000 ppm BHP in their drinking water for 12 wk and maintained without further treatment until they were sacrificed at 25 wk. A total of nine lung adenocarcinomas were obtained and total RNA was extracted to assess expression by real-time quantitative reverse transcription (RT)-polymerase chain reaction (PCR). Five out of nine adenocarcinomas showed reduced expression compared with normal lung tissue. We next performed a bisulfite sequence analysis to measure the methylation status of the 5 ' upstream region of the Cx26 gene in two normal lung tissues and five lung adenocarcinomas that showed reduced expression of Cx26. All five adenocarcinomas were highly methylated in the 5 ' upstream region, while the two normal lung tissues were unmethylated. This suggests that aberrant methylation of the Cx26 gene may be involved in the development of lung adenocarcinomas induced by BHP in rats. (c) 2006 Wiley-Liss, Inc.
  • Kyoko Shimizu; Yumi Itsuzaki; Mariko Onishi; Hiromasa Fujii; Kanya Honoki; Toshifumi Tsujiuchi
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 347 1 358 - 362 2006年08月 [査読有り]
     
    TSLC1 gene inactivation due to promoter methylation has been reported in several human cancers. Here, we investigated the expression of the Tslc1 gene and its methylation pattern in lung adenocarcinomas induced by N-nitrosobis(2-hydroxypropyl)amine (BHP). Six-week-old male Wistar rats were given 2000 ppm BHP in their drinking water for 12 weeks and maintained without further treatment until they were sacrificed at 25 weeks. Total RNA was extracted from a total of I I lung adenocarcinomas and their Tslc1 gene expressions were analyzed by real-time quantitative reverse transcription-polymerase chain reaction. Tslc1 expression was significantly reduced in the lung adenocarcinomas compared with three normal lung tissues (p < 0.05). Bisulfite sequence analysis of four lung adenocarcinomas and two normal lung tissues revealed that the 5' upstream region of the Tslc1 gene was highly methylated in the four lung adenocarcinomas, but unmethylated in the two normal lung tissues. These results suggest that aberrant Tslc1 gene methylation may be involved in BHP-induced development of lung adenocarcinomas in rats. (c) 2006 Elsevier Inc. All rights reserved.
  • K Shimizu; T Kitahashi; H Fujii; M Tsutsumi; T Mori; K Honoki; T Tsujiuchi
    ONCOLOGY REPORTS 16 1 85 - 89 2006年07月 [査読有り]
     
    Alterations of the Smad4 gene, identified as a mediator of the transforming growth factor-beta pathway, were investigated in hamster pancreatic duct adenocarcinomas (PDAs) and established cell lines. Female Syrian golden hamsters received 70 mg/kg of N-nitrosobis(2-oxopropyl)amine (BOP) followed by repeated exposure to an augmentation pressure regimen consisting of a choline-deficient diet combined with DL-ethionine then L-methionine and a further administration of 20 mg/kg BOP. A total of 12 PDAs obtained 10 weeks after beginning the experiment and three cell lines established from subcutaneously transplantable PDAs in syngeneic hamsters were examined for mutations using reverse transcription-polymerase chain reaction-single strand conformation polymorphism (RT-PCR-SSCP) analysis. A mutation was detected in only one PDA (1/12, 8.3%) in the form of an ACC to ATC (Thr to IIe) transition at codon 73; none were detected in the three cell lines. No reduced or increased expression of the Smad4 gene was detected in any case using real-time quantitative RT-PCR. These results suggest that the Smad4 gene might play a role in limited fraction of BOP-induced pancreatic duct carcinogenesis in hamsters.
  • K Shimizu; A Kato; D Hinotsume; M Shigemura; M Hanaoka; Y Shimoichi; K Honoki; T Tsujiuchi
    CANCER LETTERS 236 2 186 - 190 2006年05月 [査読有り]
     
    To clarify the involvement of the Foxp1 and Rassf1a genes in lung carcinogenesis, we investigated their expressions in lung adenocarcinomas induced by N-nitrosobis(2-hydroxypropyl)amine (BHP) in rats. Six week old male Wistar rats were given 2000 ppm BHP in their drinking water for 12 weeks and maintained without further treatment until they were sacrificed at 25 weeks. A total of 10 lung adenocarcinomas were obtained, along with the total RNA from each for assessment of expression by reverse transcription (RT)-polymerase chain reaction (PCR). The reduced expressions of the Foxp1 and Rassf1a genes were observed in some of the lung adenocarcinomas. These analyses were also confirmed by real-time quantitative RT-PCR. These results suggest that reduced expressions of Foxp1 and Rassf1a genes may play a role in the development of lung adenocarcinomas induced by BHP in rats. (c) 2005 Elsevier Ireland Ltd. All rights reserved.
  • K Shimizu; A Kato; M Shigemura; H Fujii; K Honoki; T Tsujiuchi
    MOLECULAR CARCINOGENESIS 45 2 112 - 117 2006年02月 [査読有り]
     
    To clarify the involvement of the Rassf1a gene in lung carcinogenesis, we investigated the methylation status in the 5' upstream region of the RAS-association domain family 1, isoform A (Rassf1a) gene in rat lung adenocarcinomas induced by N-nitrosobis(2-hydroxypropyl)amine (BHP). Six-week-old male Wistar rats were given 2000 ppm BHP in their drinking water for 12 wk and maintained without further treatment until they were sacrificed at 25 wk. A total of 15 lung adenocarcinomas were obtained and total RNAs were extracted from each for assessment of expression of the Rassf1a gene by reverse transcription (RT)-polymerase chain reaction (PCR) analysis. To measure the methylation status of the Rassf1a gene, five adenocarcinomas with a marked reduction of the Rassf1a expression and two normal lung tissues were used for a bisulfite sequencing analysis. While the normal lung tissue was unmethylated, all five adenocarcinomas were highly methylated in the 5' upstream region. Genomic DNAs were also extracted from 15 adenocarcinomas, and mutation analysis of the Rassf1a was performed with PCR-single-strand conformation polymorphism (SSCP) analysis. No mutations were detected throughout exons 1-6. The present results suggest that the aberrant methylation may be involved in the inactivation of the Rassf1a gene in the development of lung adenocarcinomas induced by BHP in rats. (c) 2005 Wiley-Liss, Inc.
  • A Kato; K Shimizu; Y Shimoichi; H Fujii; K Honoki; T Tsujiuchi
    MOLECULAR CARCINOGENESIS 45 2 106 - 111 2006年02月 [査読有り]
     
    The study aimed to assess the involvement of aberrant DNA methylation in lung carcinogenesis by measuring expressions of E-cadherin and p16 genes, and their DNA methyation status in the 5' upstream region in rat lung adenocarcinomas induced by N-nitrosobis(2-hydroxypropyl)amine (BHP). Six-week-old male Wistar rats were given 2000 ppm BHP in their drinking water for 12 wk, and maintained without further treatment until they were sacrificed at 25 wk. A total of nine lung adenocarcinomas were collected, and total RNA from each sample was extracted for assessment of gene expression by real-time reverse transcription (RT)-polymerase chain reaction (PCR). Expressions of E-cadherin and p 16 genes were significantly reduced in lung adenocarcinomas (P < 0.05 and P < 0.005, respectively) compared with normal lung tissues. For methylation analysis, bisulfite sequencing was performed using two normal lung tissues and five tumors. The two normal lung tissues were all unmethylated in regions of E-cadherin and p16. In contrast, five adenocarcinomas were highly methylated, and these aberrant methylation patterns correlated with reduced expressions of E-cadherin and p16. These results suggest that aberrant DNA methylation of E-cadherin and p16 genes may play important roles in development of lung adenocarcinomas induced by BHP in rats. (c) 2005 Wiley-Liss, Inc.
  • K Shimizu; M Hanaoka; A Kato; H Fujii; K Honoki; T Tsujiuchi
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 336 1 49 - 53 2005年10月 [査読有り]
     
    The expression of the E-cadherin gene and its DNA methylation status in the 5' upstream region were investigated in pancreatic duct adenocarcinomas (PDAs) induced by N-nitrosobis(2-oxopropyl)amine (BOP) in hamsters. Female Syrian golden hamsters received 70 mg/kg BOP, followed by repeated exposure to an augmentation pressure regimen consisting of a choline-deficient diet combined with DL-ethionine then L-methionine and a further administration of 20 mg/kg BOP. A total of 15 PDAs were obtained, along with total RNA for assessment of expression by real-time quantitative reverse transcription-polymerase chain reaction. The expression of the E-cadherin was significantly reduced in PDAs (p < 0.05) compared with normal pancreatic tissue. For the analysis of methylation status, bisulfite sequencing was performed with two normal pancreatic tissues and six tumors. The normal pancreatic tissue was all demethylated in this region of E-cadherin. In contrast, six PDAs were highly methylated, correlating with reduced expression of the E-cadherin. These results suggest that aberrant DNA methylation of the E-cadherin gene may play a role in the development of PDAs induced by BOP in hamsters. (c) 2005 Elsevier Inc. All rights reserved.
  • M Hanaoka; K Shimizu; M Shigemura; A Kato; H Fujii; K Honoki; T Tsujiuchi
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 333 4 1249 - 1253 2005年08月 [査読有り]
     
    The hamster model of pancreatic carcinogenesis is useful for understanding the development of human pancreatic cancer. However; there is only a small amount of hamster genetic information available for analyzing the gene alterations in hamster pancreatic cancers. Here, we determined the nucleotide sequence of the 5' upstream region of the hamster p16 gene using a suppression polymerase chain reaction method combined with gene-specific primers. Based on this sequence, we analyzed the methylation status of the 5' region by bisulfite sequencing in three normal pancreatic tissues and five pancreatic duct adenocarcinomas (PDAs). All five PDAs were highly methylated in the 5' upstream region and showed reduced expressions of the p16 gene, while the three normal samples were demethylated. The method described in this study is a highly effective and rapid technique for determining the 5' upstream region, and is applicable to epigenetic studies of the methylation status of this region. (c) 2005 Elsevier Inc. All rights reserved.
  • T Tsujiuchi; T Mori; T Amanuma; N Tanaka; M Tsutsumi
    CANCER LETTERS 217 1 97 - 103 2005年01月 [査読有り]
     
    To investigate characteristic of lung adenocarcinoma growth behavior, we have established a cell line (rat lung cancer in Nara (RLCNR)) from a tumor induced by N-nitrosobis(2-hydroxypropyl)amine (BHP) in a rat. This clone shows an epithelial cell morphology and grows as sheets in culture with an approximate doubling time of 19.2 h. Ultrastructurally, the RLCNR contains lamellar bodies in cytoplasm and the microvilli are present at the free cell surfaces. The line features well-developed desmosomes. The modal chromosome number of 42 is the same as for normal rat cells and its frequency was established to be 80.5%. To evaluate tumorigenicity, appropriate numbers of the cells were transplanted into syngeneic rats, but no tumor formation occurred. Genetic analyses revealed the RLCNR to have a GGT to GAT mutation at codon 12 of Ki-ras, but no p53 alteration. p16 gene expression was lost, associated with hypemethylation of CpG sites in the 5' upstream region of the gene. These results indicate that the present newly established cell line originated from an alveolar type 11 lesion of the lung. It should be useful for further investigation of in vivo growth mechanisms, especially tumor progression, of lung adenocarcinomas, since it has low malignant potential. (C) 2004 Elsevier Ireland Ltd. All rights reserved.
  • T Kitahashi; T Tsujiuchi; K Satoh; K Ohtsuki; Y Konishi; M Tsutsumi
    EXPERIMENTAL AND TOXICOLOGIC PATHOLOGY 56 3 153 - 157 2004年12月 [査読有り]
     
    Aberrant transcription of the fragile histidine triad (FHIT) gene was investigated in intrahepatic cholangiocellular carcinomas (ICCs) induced by N-nitrosobis(2-oxopropyl)amine (BOP) in female Syrian golden hamsters. The animals received 70 mg/kg of BOP followed by repeated exposure to an augmentation pressure regimen consisting of a choline-deficient diet combined with DL-ethionine and then L-methionine and administration of 20 mg/kg BOP. A total of 14 ICCs were obtained 10 weeks after the beginning of the experiment and total RNAs were extracted from each for assessment of aberrant transcription of the FHIT gene by reverse transcription-polymerase chain reaction analysis. Aberrant transcripts were detected in four out of 14 ICCs (28.6%), as absence in the regions of nucleotides (nt) -75 to 279, nt -75 to 348 and nt -75 to 447. These results suggest that alteration of the FHIT gene may play a role in a small fraction of ICCs induced by BOP in the hamster. (C) 2004 Elsevier GmbH. All rights reserved.
  • T Tsujiuchi; Y Sasaki; Y Oka; H Kuniyasu; M Tsutsumi
    MOLECULAR CARCINOGENESIS 40 4 241 - 246 2004年08月 [査読有り]
     
    Abnormalities of tumor suppressor genes (TSGs) on chromosome 3p are known to be important for the development of human lung cancers. In the present study, we investigated alterations of the Dutt1/Robo1 gene, as a possible tumor suppressor in this region, in rat lung adenocarcinomas induced by N-nitrosobis(2-hydroxypropyl)amine (BHP). Male Wistar rats, 6-wk-old, were given 2000 ppm BHP in their drinking water for 12 wk and maintained without further treatment until killed at wk 25. A total of 12 lung adenocarcinomas were obtained and total RNAs were extracted from each for assessment of aberrant transcripts of the Dutt1/Robo1 gene by reverse transcription (RT)-polymerase chain reaction (PCR) analysis. Aberrant transcripts bearing deletions of nucleotides (nt) 55-4318, 894346, 605-4221, and 929-4318 were detected in four of 12 adenocarcinomas (33.3%). Loss or reduced expression of the Dutt1/Robo1 gene was not found in any of the adenocarcinomas. Genomic DNAs extracted from six adenocarcinomas for Southern blot analysis did not show any evidence of deletion or gross rearrangement of the Dutt1/Robo1 gene. These results suggest that alterations of the Dutt1/Robo1 gene may be involved in the development of some lung adenocarcinomas induced by BHP in rats. (C) 2004 Wiley-Liss, Inc.
  • K Honoki; K Yoshitani; T Tsujiuchi; T Mori; M Tsutsumi; T Morishita; Y Takakura; Y Mii
    ONCOLOGY REPORTS 11 5 1025 - 1030 2004年05月 [査読有り]
     
    Flavopiridol is the potent inhibitor of cdks sharing its function with endogenous cdk inhibitors, and causes arrest at both the G1 and G2 phases of the cell cycle resulting in apoptosis in various tumor cell lines. Cyclin-dependent kinase inhibitor p16(INK4a) induces cell cycle arrest in G1 or G2 or both, and is inactivated in many malignant tumors. In this study, we focused on the effects of flavopiridol on chemically-induced rat lung adenocarcinoma, osteosarcoma and malignant fibrous histiocytoma (MFH) cell lines showing different pattern of p16(INK4a) status. The data demonstrated that flavopiridol inhibited cellular growth in a dose- and time-dependent manner, inducing apoptosis within 24 h in all cell lines at a concentration of 300 nM. The growth inhibition rate was the greatest for lung adenocarcinoma cells, lacking p16(INK4a) expression associated with methylation-mediated gene silencing; 83% at a concentration of 300 nM for 72-h treatment; while the growth of osteosarcoma and MFH cells, both expressing p16(INK4a), were inhibited at similar levels; 54-61% for osteosarcoma and 61-64% for MFH cell lines. Then, we further investigated the influence of p16(INK4a) induction upon the effect of flavopiridol in p16(INK4a)-deficient lung adenocarcinoma cells. 5-aza 2'-deoxycytidine (5-Aza-CdR) induced p16(INK4a) expression and inhibited cellular growth in lung adenocarcinoma at a similar level to that with flavopiridol treatment. After the induction of p16(INK4a) expression by 5Aza-CdR, the growth inhibition rates of flavopiridol in the p16(INK4a)-induced lung adenocarcinoma cells could not achieve comparable inhibition to that in the p16(INK4a)-deficient cells; the efficacy was reduced compared to original p16(INK4a)-deficient cells at each concentration of 50, 100 and 500 nM for 72-h treatment. These data indicate that flavopiridol shows cell type specific inhibition and possibly acts in a more compensatory manner for endogenous p16(INK4a) function in tumor cells having the aberrations of p16(INK4a) gene.
  • T Tsujiuchi; Y Sasaki; Y Oka; H Kuniyasu; Y Konishi; M Tsutsumi
    MOLECULAR CARCINOGENESIS 39 4 199 - 205 2004年04月 [査読有り]
     
    To elucidate whether the M6p/Igf2 receptor (M6p/Igf2r) gene might be involved in exogenous and endogenous liver carcinogenesis, we investigated its alteration in hepatocellular carcinomas (HCCs) induced by N-nitrosodiethylamine (DEN) and by a choline-deficient L-amino acid-defined (CDAA) diet in rats. Male F344 rats, 6 wk old, received a single intraperitoneal (i.p.) injection of DEN at a dose of 10 mg/kg body weight, followed by combined treatment with partial hepatectomy and colchicine to induce cell cycle disturbance, and a selection procedure regimen, HCCs being obtained after 42 wk. With continuous CDAA diet feeding, tumors were sampled after 75 wk. Total RNA was extracted from individual HCCs for assessment of mutations within exons 27, 28, 31, 33, and 34, and aberrant transcript of the M6p/ Igf2r gene by reverse transcription (RT)-polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP) and RT-PCR analyses, respectively. Mutations were detected in three of 15 HCCs (20%) induced by the CDAA diet, a TTT to TTG (Phe to Leu) transversion at codon 1516 and two AAG to AGG (Lys to Arg) transitions at codon 1620, but in none of those caused by DEN. Aberrant transcripts were found in seven of 15 HCCs after DEN treatment (46.7%) and in two of 15 HCCs induced by the CDAA diet (13.3%). These results suggest that alterations of the M6p/ Igf2r gene may be involved in both exogenous and endogenous liver carcinogenesis with the different patterns and frequencies. (C) 2004 Wiley-Liss, Inc.
  • K Honoki; T Tsujiuchi; T Mori; K Yoshitani; M Tsutsumi; Y Takakura; Y Mii
    MOLECULAR CARCINOGENESIS 39 1 10 - 14 2004年01月 [査読有り]
     
    Loss of p16(INK4a) protein expression has frequently been related to DNA methylation in association with gene silencing. Although the methylation status of exon1alpha for p16(INK4a) involvement in various cancers has been extensively analyzed, it has been pointed out that some inconsistencies existed in its relationship to gene silencing of p16(INK4a). In this study, we focused on the expression and methylation status in the regions of nt -478 to -201, containing a putative TATA box (nt -401 to -396), and nt -233 to 26, both in a recently cloned 5' upstream region of rat p16(INK4a). We showed that rat lung adenocarcinoma RLCNR did not express the p16(IK4a) gene, whereas rat osteosarcoma COS1NR and malignant fibrous histiocytoma MFH1NR both expressed it at levels similar to normal fibroblasts, even though the region of nt -233 to 26 was hypermethylated in COS1NR rather than RLCNR. In contrast, the CpG islands near the putative TATA box region were consistently methylated in RLCNR, but not in COS1NR and MFH1NR, as well as in normal fibroblasts. Treatment with 5-aza 2'-deoxycytidine induced expression of p16(INK4a) gene in RLCNR after 48 h, but no changes were observed in COS1NR and MFH1NR. The results indicated that methylation of CpG islands near a TATA box region played a critical role for gene silencing of the rat p16(INK4a) gene, rather than that of other regions. (C) 2003 Wiley-Liss, Inc.
  • T Tsujiuchi; Y Sasaki; Y Oka; Y Konishi; M Tsutsumi
    MOLECULAR CARCINOGENESIS 36 3 147 - 152 2003年03月 [査読有り]
     
    Alterations of the fragile histidine triad (Fhit) gene were investigated in rat hepatocarcinogenesis induced by a choline-deficient L-amino acid-defined (CDAA) diet. Males of the F344 strain, 6 wk of age, were fed a CDAA diet, and subgroups were killed at 2, 4, 12, 20, and 75 wk after the beginning of the experiment. Fifteen hepatocellular carcinomas (HCCs) were noted in rats by the last time point; they were dissected free from the surrounding tissue. Normal control liver specimens were obtained from 6-wk-old rats. Total RNAs were extracted from whole livers of animals fed the CDAA diet for 2, 4, 12, and 20 wk and from HCCs, for assessment of aberrant transcription of the Fhit gene by reverse transcription-polymerase chain reaction. Aberrant transcripts were detected in livers of rats fed the CDAA diet for 4, 12, and 20 wk, but not 2 wk, as well as in 11 of 15 HCCs (73.3%). Southern blot analysis showed a genomic DNA abnormality in one of seven informative HCCs (14.3%), while Western blot analysis showed reduction of Fhit protein expression in seven of nine HCCs (77.8%). No abnormal expression was evident in the livers after exposure to the CDAA diet for 2-20 wk. These results suggest that Fhit alterations may play important roles in hepatocarcinogenesis due to choline deficiency in rats. (C) 2003 Wiley-Liss, Inc.
  • T Tsujiuchi; Y Sasaki; T Kubozoe; Y Konishi; M Tsutsumi
    MOLECULAR CARCINOGENESIS 36 2 60 - 66 2003年02月 [査読有り]
     
    Alteration of the Fhit gene was investigated in pancreatic duct adenocarcinomas induced by N-nitrosobis-(2-oxopropyl)amine (BOP) in Syrian golden hamsters. The animals received 70 mg/kg BOP, followed by repeated exposure to an augmentation pressure regimen consisting of a choline-deficient diet combined with DL-ethionine and then L-methionine and administration of 20 mg/kg BOP. A total of 15 pancreatic duct adenocarcinomas were obtained 10 wk after the beginning of the experiment, and total RNAs were extracted from each for assessment of aberrant transcription of the Fhit gene by reverse transcription-polymerase chain reaction analysis. Aberrant transcripts lacking nucleotides in the regions of nt -75 to 348, nt -15 to 348, or nt -75 to 178 were detected in 11 adenocarcinomas (73.3%). Southern blot analysis of eight tumors did not show any evidence of gross rearrangement or deletion. These results indicated that changes in the Fhit gene occurred frequently and thus may have played a role in the development of pancreatic duct adenocarcinomas induced by BOP in hamsters. (C) 2003 Wiley-Liss, Inc.
  • T Tsujiuchi; Y Sasaki; M Tsutsumi; Y Konishi
    MOLECULAR CARCINOGENESIS 36 1 32 - 37 2003年01月 [査読有り]
     
    Alterations of the mannose 6-phosphate/insulin-like growth factor II receptor (M6p/lgf2r) gene were investigated in lung adenocarcinomas induced by N-nitrosobis(2-hydroxypropyl)amine (BHP) in rats. Male Wistar rats, 6 wk old, were given 2000 ppm BHP in their drinking water for 12 wk and maintained without further treatment until killed at week 25. A total of 12 lung adenocarcinomas were obtained, and total RNAs were extracted from each for assessment of mutations and levels of aberrant transcripts of the M6p/1gf2r gene by reverse transcription (RT)-polymerase chain reaction (PCR) single-strand conformation polymorphism analysis and RT-PCR analysis, respectively. No mutations were found in exons 27, 28, 31, 33, and 34. Aberrant transcripts bearing deletions of hit 3698 to 4902, 3366 to 4902, and 3817 to 4697 were detected in three of 12 adenocarcinomas (25%). These results suggest that alterations of the M6p/1gf2r gene may be involved in the development of lung adenocarcinomas induced by BHP in rats. (C) 2002 Wiley-Liss, Inc.
  • Tsujiuchi T; Tsutsumi M; Konishi Y
    J Toxicol Pathol 16 133 - 138 2003年 [査読有り]
  • T Tsujiuchi; Y Sasaki; Y Konishi; M Tsutsumi
    MOLECULAR CARCINOGENESIS 35 2 57 - 62 2002年10月 [査読有り]
     
    Alteration of the retinoblastoma-related gene RB2/p130 was investigated in lung adenocarcinomas induced by N-nitrosobis(2-hydroxypropyl)amine (BHP) in male Wistar rats. At 6 wk of age, 21 animals were given 2000 ppm of BHP in their drinking water for 12 wk and then maintained without further treatment until they were killed at the end of week 25. A total of 21 lung adenocarcinomas were obtained, and total RNAs were extracted from each for mutation analysis of R92/p130 by the reverse transcription-polymerase chain reaction-single-strand comformation polymorphism approach. No mutations were found in exons 19-22. However, examination of the expression of the RB2/p130 gene by Northern blot analysis showed mRNA levels to be significantly lower than those of normal lung tissues. Western blot analysis showed reduction of the pRb2/p130 protein in all of the adenocarcinomas examined. These results suggest that alteration of the RB21p130 gene may play important roles in the development of lung adenocarcinomas induced by BHP in rats. (C) 2002 Wiley-Liss, Inc.
  • T Tsujiuchi; Y Sasaki; T Kubozoe; M Tsutsumi; Y Konishi; D Nakae
    MOLECULAR CARCINOGENESIS 34 1 19 - 24 2002年05月 [査読有り]
     
    The present study was conducted to assess whether Fhit gene alterations are a feature of hepatocellular carcinomas (HCCs) induced by N-nitrosodiethylamine (DEN) in male Fischer 344 rats. Animals, 6 wk old, received a single intraperitoneal injection of DEN at a dose of 10 mg/kg body weight, followed by combined treatment with partial hepatectomy and colchicine to induce cell-cycle disturbance and a selection procedure, consisting of 2-acetylaminofluorene and carbon tetrachloride. Fourteen HCCs were obtained 42 wk after the beginning of the experiment; total RNA was extracted for the assessment of aberrant transcription of the Fhit gene by reverse transcriptase-polymerase chain reaction analysis. Aberrant transcripts were detected in nine of the 14 HCCs (64.3%). Sequence analysis showed that these resulted from the absence of nt -9 to 279, nt -9 to 348, nt -98 to 279, nt -26 to 365, or nt -98 to 348. Western blot analysis demonstrated reduced expression of Fhit protein in six of 10 HCCs (60.0%), with a perfect correlation with Fhit gene alterations, These results indicated that changes in the Fhit gene occur frequently and may thus play some role in the development of HCCs induced by DEN in rats. (C) 2002 Wiley-Liss, Inc.
  • K Honoki; T Tsujiuchi; Y Sasaki; M Tsutsumi; T Morishita; A Kido; Y Miyauchi; Y Mii; Y Takakura; Y Konishi
    MOLECULAR CARCINOGENESIS 33 2 81 - 87 2002年02月 [査読有り]
     
    Cytokines are considered to play an important role in tumor pathogenesis and progression, and recent studies have demonstrated that a variety of forms, including interleukins (ILs) and transforming growth factor-betas (TGF-betas), may regulate tumors. In the present study, the expression of TGF-beta isoforms and ILs was investigated in cell lines from a rat osteosarcoma and a malignant fibrous histiocytoma (MFH), both established from transplantable tumors induced by 4(hydroxyamino) quinoline 1-oxide (4-HAQO) in syngeneic F344 male rats. The results of a multiprobe RNase protection assay showed TGF-beta1 expression to be remarkably elevated, with no TGF-beta2 and beta3 detectable in MFH cells, while TGF-beta1 and -beta2 were found to be moderately and TGF-beta3 weakly expressed in osteosarcoma lines. All cell lines of osteosarcomas and MFHs expressed macrophage migration inhibitory factor at similar levels. In contrast to the lack of ILs in the MFH cells, moderate IL-6 and very weak IL-1beta expression was detected in the osteosarcoma cells. These results suggest that variation in expression pattern of these cytokines in osteosarcomas and MFHs might be involved in differences in histological appearance and biological behavior, including metastatic ability, between these two mesenchyme-derived tumor types. (C) 2002 Wiley-Liss, Inc.
  • A Denda; WA Kitayama; H Kishida; N Murata; M Tsutsumi; T Tsujiuchi; D Nakae; Y Konishi
    JAPANESE JOURNAL OF CANCER RESEARCH 93 2 125 - 132 2002年02月 [査読有り]
     
    Development of hepatocellular carcinomas in rats caused by a choline-deficient, L-amino acid-defined (CDAA) diet, usually associated with fatty liver, fibrosis, cirrhosis and oxidative DNA damage, has been recognized as a useful model of hepatocarcinogenesis caused by endogenous factors. In the present study, in order to further explore involved factors and genes, we established an equivalent model in spontaneous liver tumor-resistant C57BL/6J mice. Six-week-old males and females were continuously fed the CDAA diet and histological liver lesions and oxidative DNA damage due to 8-hydroxydeoxyguanosine (8-OHdG) were examined after 22, 65 and 84 weeks. In male mice, fatty change and fibrosis were evident at 22 weeks, and preneoplastic foci of altered hepatocytes were seen at an incidence of 8/8 (100%) and a multiplicity of 6.6 +/- 4.0 per mouse at 65 weeks. Hepatocellular adenomas and carcinomas developed at incidences of 16/24 (66.7%) and 5/24 (20.8%), and multiplicities of 1.42 +/- 1.32 and 0.29 +/- 0.62, respectively, at 84 weeks. The female mice exhibited resistance to development of these lesions. The CDAA diet also increased 8-OHdG levels in male but not female mice. These results indicate that a CDAA diet causes hepatocellular preneoplastic foci, adenomas and carcinomas associated with fibrosis and oxidative DNA damage in mice, as in rats, providing a hepatocarcinogenesis model caused by endogenous factors in mice.
  • A Denda; W Kitayama; A Murata; H Kishida; Y Sasaki; O Kusuoka; T Tsujiuchi; M Tsutsumi; D Nakae; H Takagi; Y Konishi
    CARCINOGENESIS 23 2 245 - 256 2002年02月 [査読有り]
     
    Expression of cyclooxygenase (COX)-2 protein during rat hepatocarcinogenesis associated with fatty change, fibrosis, cirrhosis and oxidative DNA damage, caused by a choline-deficient, L-amino acid-defined (CDAA) diet were investigated in F344 male rats, along with the chemopreventive efficacy of the specific COX-2 inhibitor, nimesulide (NIM). Nimesulide, which was administered in the diet at concentrations of 200, 400, 600 and 800 p.p.m. for 12 weeks, decreased the number and size of preneoplastic enzyme-altered liver foci, levels of oxidative DNA damage, and the grade and incidence of fibrosis in a dose-dependent manner. A preliminary long-term study of 65 weeks also revealed that 800 p.p.m. NIM decreased the multiplicity of neoplastic nodules and hepatocellular carcinomas and prevented the development of cirrhosis. Western blot analysis revealed that COX-2 protein was barely expressed in control livers and increased similar to2.9-fold in the livers of rats fed on a CDAA diet for 12 weeks and similar to4.5-5.4-fold in tumors, with a diameter larger than 5 mm, at 80 weeks. Immunohistochemically, COX-2 protein was positive in sinusoidal and stromal cells in fibrotic septa, which were identified by immunoelectron microscopy as Kupffer cells, macrophages, either activated Ito cells or fibroblasts, after exposure to the CDAA diet for 12 weeks, whereas it was only occasionally weakly positive in sinusoidal, probably Kupffer, cells in control livers. In neoplastic nodules in rats fed on a CDAA diet for 30 and 80 weeks, sinusoidal cells and cells with relatively large round nuclei and scanty cytoplasm were strongly positive for COX-2 protein, with the neoplastic hepatocytes in the minority of the nodules, but not the cancer cells, being moderately positive. These results clearly indicate that rat hepatocarcinogenesis, along with fatty change, fibrosis and cirrhosis, is associated with increased expression of COX-2 protein, and point to the chemopreventive efficacy of a selective COX-2 inhibitor against, at least, the early stages of hepatocarcinogenesis.
  • Tadahiko Kubozoe; Masahiro Tsutsumi; Nao Murata; Toshifumi Tsujiuchi; Tsukasa Tsunoda; Takashi Sugimura; Keiji Wakabayashi; Yoichi Konishi
    Journal of Toxicologic Pathology 15 1 7 - 12 2002年 [査読有り]
     
    The carcinogenic potential of 9-(4'-aminophenyl)-9H-pyrido[3,4-e]indole (aminophenylnorharman: APNH), a newly identified heterocyclic amine, was evaluated in a rapid production model for pancreatic duct adenocarcinoma in hamsters. In experiment 1, the initiation potential of APNH was examined. Hamsters received corn oil, 30 mg/kg body weight N-nitrosobis(2-oxopropyl)amine (BOP) or APNH (totally 50 and 100 mg/kg) as initiators followed by two cycles of augmentation pressure for pancreatic duct carcinogenesis and were sacrificed 75 to 78 days after the beginning of the experiment. Ductal hyperplasia and atypical hyperplasias developed in hamsters receiving APNH but their numbers and incidences did not differ from the corn oil case. In experiment 2, promoting potential of APNH for pancreatic duct carcinogenesis was examined. Hamsters received 30 mg/kg body weight BOP as an initiator followed by augmentation pressures and thereafter diet containing 0, 25 or 50 ppm APNH for 50 days. All hamsters were sacrificed 100 days after the beginning of the experiment. The numbers and incidences of ductal lesions, including adenocarcinomas, did not differ among the experimental groups. The results suggest, APNH may not have initiation or promotion potential for pancreatic duct carcinogenesis in hamsters.
  • T Tsujiuchi; Y Sasaki; N Murata; M Tsutsumi; Y Konishi; D Nakae
    CARCINOGENESIS 22 12 2017 - 2022 2001年12月 [査読有り]
     
    Alteration of the FHIT gene was investigated in lung adenocarcinomas induced by N-nitrosobis(2-hydroxypropyl) amine (BHP) in male Wistar rats. Animals at 6 weeks of age were given 2000 p.p.m. of BHP in drinking water for 12 weeks, then maintained without further treatment until killed at the end of week 25. A total of 25 lung adenocarcinomas were obtained and total RNAs were extracted from each for assessment of aberrant transcription of the FHIT gene by reverse transcription (RT)-polymerase chain reaction (PCR) analysis. Aberrant transcripts were detected in 15 adenocarcinomas (60%) as absence in the regions of nucleotides (nt) -9 to 279, -98 to 279, -98 to 348 or -98 to 447. Genomic DNAs were also extracted from all 25 adenocarcinomas and exons 5-9 were examined for mutations, using PCR-single strand conformation polymorphism (SSCP) analysis and sequencing. A mutation was detected in only one adenocarcinoma (4%), an ACC to ATC (Thr to IIe) transition at codon 76. Southern blot analysis of eight tumors did not show any evidence of gross rearrangement or deletion of the FHIT gene. Western blot analysis revealed reduced expression of Fhit protein in six out of 10 adenocarcinomas (60%). These results suggest that alteration of the FHIT gene may be involved in the development of lung adenocarcinomas induced by BHP in rats.
  • T Tsujiuchi; Y Sasaki; N Murata; M Tsutsumi; D Nakae; Y Konishi
    EXPERIMENTAL AND TOXICOLOGIC PATHOLOGY 53 4 291 - 295 2001年09月 [査読有り]
     
    Expression of transforming growth factor Ps (TGF Ps), tumor necrosis factor (TNF) family members, interferons (IFNs), macrophage migration inhibitory factor (NEF) and granulocyte macrophage colony stimulating factor (GMCSF) in lung adenocarcinomas induced by N-nitrosobis(2-hydroxypropyl)amine (BHP) in rats was investigated using a multiprobe RNase protection assay (RPA) followed by densitometric quantification. Male Wistar rats, 6 weeks of age, were given 2000 ppm BHP in their drinking water for 12 weeks and maintained without further treatment until killed at week 25. Total RNAs were extracted from 15 adenocarcinomas. Four samples of normal lung tissue from untreated rats served as controls. The expression of TGF beta1, TGF beta2, TGF beta3, TNF alpha, TNF beta and lymphotoxin P (Ltp) was significantly higher in adenocarcinomas than in normal lung tissues. In contrast, MIF was expressed at the same level in neoplasms and normal tissue and no expression of IFN beta, IFN gamma and GM-CSF was apparent in either adenocarcinomas or normal lung tissues. These results suggest that elevated expression of TGF betas and TNF family members may contribute to the development and progression of lung adenocarcinomas induced by BHP in rats.
  • TO Kubozoe; T Tsujiuchi; N Murata; Y Sasaki; T Tsunoda; Y Konishi; R Tsutsumi
    CANCER LETTERS 168 1 1 - 6 2001年07月 [査読有り]
     
    The involvement of beta -catenin gene alterations in pancreatic duct carcinogenesis induced by N-nitrosobis(2-oxopropyl)amine (BOP) in hamsters was examined by polymerase chain reaction-single strand conformation polymorphism analysis and the expression of beta -catenin protein was examined by immunohistochemistry. No mutations of the beta -catenin gene were detected in 20 pancreatic duct adenocarcinomas (PDAs). Immunohistochemical staining showed the beta -catenin protein to be ubiquitously localized in the cell membranes, beta -Catenin accumulation was not identified in the cytoplasm and/or nucleus in any of 102 hyperplasias, 35 atypical hyperplasias, and 73 PDAs, as well as normal pancreatic duct cells. These results suggest that the Wnt/beta -catenin signaling pathway may not play an important role in pancreatic duct carcinogenesis induced by BOP in hamsters. (C) 2001 Elsevier Science ireland Ltd. All rights reserved.
  • T Tsujiuchi; Y Sasaki; M Tsutsumi; Y Konishi
    CANCER LETTERS 162 2 207 - 211 2001年01月 [査読有り]
     
    Mutations of the phosphatase and tensin homolog deleted on chromosome ten/mutated in multiple advanced cancers 1 (PTEN/MMAC1) gene were investigated in lung adenocarcinomas induced by N-nitrosobis(2-hydroxypropyl)amine (BHP) in rats. Male Wistar rats (6 weeks old) were given 2000 ppm BHP in their drinking water for 12 weeks and maintained without further treatment until they were killed at week 25. Total RNA was extracted from 15 adenocarcinomas and mutations of the PTEN/MMAC1 gene were investigated by reverse transcription-polymerase chain reaction-restriction-single-strand conformation polymorphism analysis. No mutations were detected, and loss or decrease of expression of PTEN/MMAC1 mRNA was also not found in any of the cases. These results suggest that alterations of the PTEN/MMAC1 gene may not be involved in the development of adenocarcinomas induced by BHP in rats. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.
  • M Tsutsumi; M Masutani; T Nozaki; O Kusuoka; T Tsujiuchi; H Nakagama; H Suzuki; Y Konishi; T Sugimura
    CARCINOGENESIS 22 1 1 - 3 2001年01月 [査読有り]
     
    The involvement of poly(ADP-ribose) polymerase-1 (Parp-1), one of the poly(ADP-ribose) polymerase family proteins, in genomic stability, DNA repair and cell death triggered by DNA damage has been well documented. However, the potential role of Parp-1 in carcinogenesis has not been well evaluated. In this study the carcinogenic activity of N-nitrosobis(2-hydroxypropyl)amine (BHP) was studied in Parp-1(-/-) mice, generated by disrupting Parp-1 gene exon 1, Parp-1(-/-) and Parp-1(+/+) male mice received 0, 250 and 500 p.p.m. BHP in their drinking water for 20 weeks and were then killed. The percentage of animals bearing hemangiomas and hemangiosarcomas in the liver and numbers of tumors per mouse were markedly higher in the Parp-1(-/-) groups given 250 or 500 p.p.m. BHP than in their Parp-1(+/+) counterparts. Hemangiosarcomas developed only in Parp-1(-/-) mice. In the lung the numbers of adenomas per mouse were increased in Parp-1(-/-) mice given BHP at 250 and 500 p.p.m. (P < 0.01) compared with the Parp-1(+/+) case. The results show that susceptibility to BHP is significantly elevated in Parp-1(-/-) mice, thus providing direct evidence that Parp-1 is relevant to carcinogenesis.
  • H Kuniyasu; N Oue; D Nakae; M Tsutsumi; A Denda; T Tsujiuchi; H Yokozaki; W Yasui
    PATHOBIOLOGY 69 2 86 - 95 2001年 [査読有り]
     
    Interleukin 15 (IL-15 mRNA expression was detected in human colorectal cancer cells (Colo320, WiDr, TCO and DLD1) by the reverse transcriptase-polymerase chain reaction (RT-PCR). Only Colo320 and WiDr cells secreted IL-15 culture medium. With IL-15 treatment, all cell lines grew at a rate of 120-180% of that of nontreated cells. A binding assay with I-125-labeled IL-15 showed binding activity to IL-15 in Colo320 (K-d: 0.098 nM) cells. IL-15 also reversed the growth inhibition caused by serum starvation in Colo320 cells. IL-15-induced cell growth in regular and serum-free media was abrogated by anti-IL-15 antibody treatment in Colo320 cells. Moreover, IL-15 treatment reduced doxorubicin-induced cytostasis and cytolysis in Colo320 cells by 50%. The invasion capacity of IL-15-treated Colo320 cells was 5.3 times that of untreated cells. Immunoblotting showed that IL-15-treated Colo320 cells exhibited downregulation of p21Waf1 and Bax, and upregulation of Bcl-2, phospho-AKT, MMP9/ MMP2, and VEGF. Finally, immunostaining of human colon cancer revealed that 33 (70%) of 47 Dukes' C cases showed IL-15 expression in cancer cells, whereas only 16% of Dukes' B cases did (p < 0.0001). IL-15 may play important roles in cell proliferation, invasion, and metastasis of human colorectal cancer. Copyright 0 2001 S. Karger AG, Basel.
  • Yoshiharu Takashima; Masahiro Tsutsumi; Yasutaka Sasaki; Toshifumi Tsujiuchi; Osamu Kusuoka; Yoichi Konishi
    Journal of Toxicologic Pathology 14 2 87 - 98 2001年 [査読有り]
     
    Safety assessment of endocrine-disrupting chemicals (EDCs) is now an important issue because of fear of human exposure. Bisphenol A (BPA), a compound initially synthesized as a chemical oestrogen, is now used as a monomer for the production of polycarbonate plastic products such as baby bottles. In the present experiment, toxicity and effects of BPA administration to maternal rats and response of their offspring to N-nitrosobis(2hydroxypropyl)amine (BHP) treatment were studied. Growth retardation was observed in maternal rats throughout the period of BPA exposure (total dose of 21 ± 3 grams per rat). Serum TSH was elevated in maternal rats receiving a soybean-devoid diet and offspring of maternal rats with or without BPA but this was not thought to be caused by BPA. Histopathologically, no significant organ toxicity was observed in BPA-treated maternal rats. Examination of cleavage of the balanopreputial gland in males and opening of the vagina in females performed on 5 to 7-week-aged offspring, demonstrated no abnormal differentiation or growth retardation. BPA exposure did not affect BHP-induced carcinogenesis in the thyroid, lung, thymus, esophagus, and liver, location, incidences, and numbers of tumors not differing in offspring born from maternal rats with or without BPA administration. These results indicate that BPA does not induce anv tissue injury for maternal or offspring rats, with no effects on target orsan carcinoeenesis of BHP in offspring.
  • Y Sasaki; T Tsujiuchi; N Murata; M Tsutsumi; Y Konishi
    JAPANESE JOURNAL OF CANCER RESEARCH 92 1 16 - 22 2001年01月 [査読有り]
     
    To elucidate involvement of the transforming growth factor-beta (TGF-beta) signaling pathway in endogenous and exogenous liver carcinogenesis, we investigated mutations of TGF-beta receptor type II (TGF-beta RII), Smad2 and Smad4 genes, and expression of TGF-beta RII in hepatocellular carcinomas (HCCs) induced by a choline-deficient L-amino acid-defined (CDAA) diet and by N-nitrosodiethylamine (DEN), Male Fischer 344 rats received a CDAA diet continuously and HCCs were sampled after 75 weeks. Administration of DEN was followed by partial hepatectomy (PH), with colchicine to induce cell cycle disturbance and a selection pressure regimen, HCCs being obtained after 32 weeks. Total RNAs were extracted from individual HCCs and mutations in TGF-beta RII, Smad2 and Smad4 were investigated by reverse transcription (RT)-polymerase chain reaction (PCR)-restriction-single-strand conformation polymorphism (SSCP) analysis followed by sequencing analysis, Mutations of Smad2 were detected in 2 out of 12 HCCs (16.7%) induced by the CDAA diet, a GGT-to-GGC transition (Gly to Gly) at codon 30 and a TCT-to-GCT (Ser to Ala) transversion at codon 118, without any TGF-beta RII or Smad4 alterations, No mutations of TGF-beta RII, Smad2 and Smad4 were encountered in eleven HCCs induced by the exogenous carcinogen. Semi-quantitative RT-PCR revealed reduced expression of TGF-beta RII in 2 HCCs (16.7%) without Smad2 mutations out of 12 HCCs induced by the CDAA diet and none of II induced by DEN, These results suggest that the TGF-beta signaling pathway may be disturbed in endogenous liver carcinogenesis in rats.
  • T Tsujiuchi; M Tsutsumi; Y Sasaki; N Murata; Y Konishi
    CANCER RESEARCH 60 23 6611 - 6616 2000年12月 [査読有り]
     
    In the present study, we investigated mutations of the adenomatous polyposis coli (APC) and beta -catenin genes to clarify possible molecular mechanisms underlying development of lung tumors induced by N-nitrosobis(2-hydroxypropyl)amine (BHP) in rats. Male Wistar rats, 6 weeks of age, were given 2000 ppm BHP in drinking water for 12 weeks and then maintained without further treatment until sacrifice at week 25, DNA was extracted from paraffin-embedded tissues, and PCR-single-strand conformation polymorphism analysis, followed by nucleotide sequencing, was performed. No APC mutations were detected in 17 hyperplasias, but 2 of 15 adenomas (13.3%) and 8 of 20 adenocarcinomas (40.0%) showed changes within exon 1 to the mutation cluster region in exon 15, For beta -catenin, no mutations were detected in 17 hyperplasias, but 3 of 15 adenomas (20.0%) and 5 of 20 adenocarcinomas (25.0%) had alterations within or flanking codons corresponding to important phosphorylation sites. Immunohistochemical staining showed beta -catenin protein localized in the cell membranes in the surrounding normal-appearing lung and 216 hyperplasias and localized mainly in the cytoplasm and/or nucleus in 10 of 37 adenomas (27.0%) and 21 of 40 adenocarcinomas (52.5%), These results suggest that the APC-beta -catenin-T-cell factor signaling pathway is involved in the acquisition of growth advantage from adenomas to adenocarcinomas in BHP-induced rat Lung carcinogenesis.
  • T Tsujiuchi; M Tsutsumi; Y Sasaki; N Murata; Y Konishi
    CANCER RESEARCH 60 23 6611 - 6616 2000年12月 
    In the present study, we investigated mutations of the adenomatous polyposis coli (APC) and beta -catenin genes to clarify possible molecular mechanisms underlying development of lung tumors induced by N-nitrosobis(2-hydroxypropyl)amine (BHP) in rats. Male Wistar rats, 6 weeks of age, were given 2000 ppm BHP in drinking water for 12 weeks and then maintained without further treatment until sacrifice at week 25, DNA was extracted from paraffin-embedded tissues, and PCR-single-strand conformation polymorphism analysis, followed by nucleotide sequencing, was performed. No APC mutations were detected in 17 hyperplasias, but 2 of 15 adenomas (13.3%) and 8 of 20 adenocarcinomas (40.0%) showed changes within exon 1 to the mutation cluster region in exon 15, For beta -catenin, no mutations were detected in 17 hyperplasias, but 3 of 15 adenomas (20.0%) and 5 of 20 adenocarcinomas (25.0%) had alterations within or flanking codons corresponding to important phosphorylation sites. Immunohistochemical staining showed beta -catenin protein localized in the cell membranes in the surrounding normal-appearing lung and 216 hyperplasias and localized mainly in the cytoplasm and/or nucleus in 10 of 37 adenomas (27.0%) and 21 of 40 adenocarcinomas (52.5%), These results suggest that the APC-beta -catenin-T-cell factor signaling pathway is involved in the acquisition of growth advantage from adenomas to adenocarcinomas in BHP-induced rat Lung carcinogenesis.
  • M Tsutsumi; K Kadomatsu; T Tsujiuchi; H Sakitani; S Ikematsu; T Kubozoe; M Yoshimoto; T Muramatsu; S Sakuma; Y Konishi
    JAPANESE JOURNAL OF CANCER RESEARCH 91 10 979 - 986 2000年10月 [査読有り]
     
    The expression of midkine (MK) was investigated in pancreatic ductal hyperplasias, atypical hyperplasias and adenocarcinomas induced by N-nitrosobis(2-oxopropyl)amine (BOP) in hamsters, and in hamster ductal adenocarcinoma cell lines (HPD-1NR, -2NR and -3NR), MK mRNA was clearly overexpressed in invasive pancreatic duct adenocarcinomas (PCs) and the three cell lines as assessed by northern blot analysis, and MK protein expression increased from ductal hyperplasia through atypical hyperplasias, intraductal carcinomas and invasive PCs by immunohistochemistry. The extent of overexpression of MK mRNA in PCs was almost the same as in hamster whole embryonic tissue. MK is reported to be a retinoid-responsive gene, but MK mRNA expression was not affected by treatment with all-trans retinoic acid (tRA) or N-(4-hydroxyphenyl)retinamide (4-HPR) in HPD-1NR cells. The results thus suggest that MK expression is involved in the development and progression of pancreatic ductal adenocarcinomas induced by BOP in hamsters, with loss of upregulation by retinoic acid.
  • T Tsujiuchi; Y Sasaki; M Tsutsumi; Y Konishi
    MOLECULAR CARCINOGENESIS 29 2 87 - 91 2000年10月 [査読有り]
     
    Mutations of the Smad2 and Smad4 genes, identified as mediators of the transforming growth factor-beta pathway, were investigated in lung adenocarcinomas induced by N-nitrosobis(2-hydroxypropyl)amine (BHP) in rats. Male Wistar rats, 6 wk old, were given 2000 ppm BHP in their drinking water for 12 wk and maintained without additional treatment until killed at week 25. Total RNA was extracted from 12 adenocarcinomas, and mutations in Smad2 and Smad4 were investigated by reverse transcription-polymerase chain reaction restriction single-strand conformation polymorphism analysis followed by sequencing analysis. in Smad2, single adenocarcinomas showed a GGC to GAC (Gly to Asp) transition at codon 100 and an AAG to GAG (Lys to Glu) transition at codon 383. In Smad4, one adenocarcinoma exhibited a TTC to CTC (Phe to Leu) transition at codon 214. These results suggest that mutations of Smad2 and Smad4 may play roles in a limited fraction of lung adenocarcinomas induced by BHP in rats. Mol. Carcinog. 29:87-91, 2000. (C) 2000 Wiley-Liss. Inc.
  • M Hashimoto; DY Wang; T Kamo; Y Zhu; T Tsujiuchi; Y Konishi; M Tanaka; H Sugimura
    AMERICAN JOURNAL OF PATHOLOGY 157 1 21 - 27 2000年07月 [査読有り]
     
    Differential display analysis of rat lung at different developmental stages identified a fragment, HG80, which appeared on embryonic day 16.5 and thereafter. A full-length cDNA derived from a cDNA library of newborn rat lung probed with HG80 was the rat counterpart of sodium-dependent phosphate transporter type IIb, and was designated rNaPi IIb, In situ hybridization showed that rNaPi IIb was expressed in type II alveolar cells, suggesting a role in the synthesis of surfactant in the alveoli. The time-dependent changes in localization of this gene in the developing lung and its possible use as a type II pneumocyte marker are discussed.
  • K Honoki; T Tsujiuchi; M Tsutsumi; A Kido; T Morishita; M Yoshimoto; Y Miyauchi; Y Mii; S Tamai; Y Konishi
    TOXICOLOGIC PATHOLOGY 28 4 575 - 579 2000年07月 [査読有り]
     
    We recently obtained 3 cloned cell lines demonstrating the p53 mutation from a lung metastatic nodule of a rat transplantable osteosarcoma. In this study, we applied wild-type p53 gene transfer to the rat osteosarcoma cells by lipofection to investigate the effects on cell growth, expression of genes such as waf1/p21, bcl-2, and bax, and nucleosomal DNA fragmentation due to apoptosis. Reconstitution of the p53 gene inhibits cellular growth, and this growth-suppressive effect is partly due to apoptosis involving bcl-2 gene suppression in this tumor type. This rat osteosarcoma model is similar in biologic behavior to human cases and thus is very suitable for further investigation of tumorigenesis and gene therapy for osteosarcoma.
  • Y Sasaki; T Tsujiuchi; N Murata; T Kubozoe; M Tsutsumi; Y Konishi
    CANCER LETTERS 152 1 71 - 77 2000年04月 [査読有り]
     
    To clarify the involvement of tumor suppressor genes in exogenous and endogenous liver carcinogenesis, alterations of p16, p21 and p53 in hepatocellular carcinomas (HCCs) induced by N-nitrosodiethylamine (DEN) and a choline deficient L-amino acid-defined (CDAA) diet in rats were investigated. Male Fischer 344 rats received DEN at 6-week of age followed by partial hepatectomy (PH), with colchicine to induce cell cycle disturbance, and a selection pressure regimen. Sacrifice was after 42 weeks. Other animals continuously received a CDAA diet for 75 weeks and were then killed. Eleven and 15 HCCs were obtained, respectively. Total RNA was extracted from and cDNA was synthesized with reverse transcriptase to allow investigation of mutations in p16, p21 and p53 by polymerase chain reaction single strand conformation polymorphism (PCR-SSCP) analysis. Expression of p16 and p21 mRNA was also analyzed by reverse transcription (RT)-PCR. The results showed no mutations or deletions of p16, p21 and p53 in any of the HCCs induced by DEN or CDAA. Loss or decrease of p16 and p21 expression were also not found, suggesting that p16, p21 and p53 alteration may not be necessary for either exogenous or endogenous liver carcinogenesis in rats. (C) 2000 Elsevier Science Ireland Ltd. Ail rights reserved.
  • W Kitayama; A Denda; J Yoshida; Y Sasaki; M Takahama; K Murakawa; T Tsujiuchi; M Tsutsumi; Y Konishi
    CANCER LETTERS 148 2 145 - 152 2000年02月 [査読有り]
     
    Expression of cyclooxygenase (COX)-2 protein in preneoplastic and neoplastic lung lesions induced by the administration of 2000 ppm of N-nitrosobis(2-hydroxypropyl)amine (BHP) in the drinking water to Wistar male rats, was examined immunohistochemically. The majority of alveolar/bronchiolar adenomas (ADs) and all adenocarcinomas (ADCs) examined, stained positive or strongly positive for COX-2, in contrast, only a minority of alveolar/bronchiolar hyperplasias demonstrated immunoreactivity and half of the squamous cell carcinomas examined, were only weakly positive. Western blotting analysis also revealed expression of COX-2 protein in the resected ADs and ADCs. These results clearly indicate up-regulated expression of COX-2 in lung neoplastic lesions, particularly ADs and ADCs, induced by BHP in rats. (C) 2000 Elsevier Science ireland Ltd. All rights reserved.
  • M Tsutsumi; H Kitada; K Shiraiwa; M Takahama; T Tsujiuchi; H Sakitani; Y Sasaki; K Murakawa; M Yoshimoto; Y Konishi
    CARCINOGENESIS 21 2 251 - 256 2000年02月 [査読有り]
     
    The effects of antibiotics and anti-inflammatory drugs on the promotion stage of lung carcinogenesis initiated with N-nitrosobis(2-hydroxypropyl)amine (BHP) in rats were investigated in two experiments with a similar protocol, In experiment 1, rats received tap water containing 2000 p,p,m, BHP for 12 weeks followed by basal diet or basal diet containing 0.02% erythromycin (EM), 0.04% ampicillin (ABPC), 1.5% sho-saiko-to, 0.02% EM plus 1.5% shosaiko-to or 0.04% ABPC plus 1.5% sho-saiko-to for 8 weeks after BHP administration. The development of adenocarcinomas (AC), squamous cell carcinomas (SqC) and adenosquamous carcinomas (ASqC) was completely inhibited in rats given ABPC plus sho-saiko-to and the numbers of lung lesions including alveolar hyperplasias, adenomas and carcinomas were decreased in rats given EM plus sho-saiko-to or ABPC plus sho-saiko-to, Neutrophil and macrophage infiltration into alveolar spaces of the lung were also markedly suppressed. In experiment 2, rats received BHP in the same manner as in experiment 1 and basal diet or basal diet containing 0.04% ABPC, 0.006% piroxicam, 0.04% ABPC plus 0.006% piroxicam and 0.04% ABPC plus 0.75% ougon for 8 weeks. The incidence and number of carcinomas, including ACs, SqCs and ASqCs were decreased in rats given ABPC plus piroxicam or ABPC plus ougon, Bacteria, mainly Escherichia coli, were detected in broncho-alveolar lavage of rats receiving BHP, The results suggest that chronic inflammation might be involved in the progression of lung carcinogenesis by BHP in rats and its suppression may therefore be useful as a chemopreventive strategy in lung cancer clinics.
  • T. Tsujiuchi; Y. Sasaki; M. Tsutsumi; Y. Konishi
    Japanese Journal of Cancer Research 91 11 1090 - 1095 2000年 [査読有り]
     
    Mutations and expression of the transforming growth factor-β receptor type II (TGF-βRII) gene were investigated in lung adenocarcinomas induced by N-nitrosobis(2-hydroxypropyl)amine (BHP) in rats. Males of the Wistar strain, 6 weeks old, were given 2000 ppm of BHP in their drinking water for 12 weeks and then maintained without further treatment until killed at week 25. Total RNA was extracted from 12 adenocarcinomas and mutations in TGF-βRII were investigated by RT-PCR-restriction-SSCP analysis followed by sequencing analysis. Two out of 12 adenocarcinomas showed band shifts, indicative of mutations (16.7%). One was a CTG-to-TTG (Leu to Leu) transition at codon 308 without amino acid alteration and the other a frameshift deletion of one of two guanines at nucleotides 1434 to 1435 (codon 477 to 478). Semi-quantitative RT-PCR analysis demonstrated significantly reduced TGF-βRII expression in adenocarcinomas, as compared with normal lung tissue. These results suggest that TGF-βRII alterations may play a role in the acquisition of growth advantage by lung adenocarcinomas induced by BHP in rats.
  • T. Tsujiuchi; Y. Sasaki; M. Tsustumi; Y. Konishi
    Jpn. J. Cancer Res. 91 10 955 - 959 2000年 [査読有り]
     
    The expression of interleukins (ILs) in lung adenocarcinomas induced by N-nitrosobis(2-hydroxypropyl)amine (BHP) in rats was investigated using a multiprobe RNase protection assay (RPA) followed by densitometric quantification, Male Wistar rats, 6 weeks old, were given 2000 ppm BHP in their drinking water for 12 weeks and maintained without further treatment until they were killed at week 25. Total RNAs were extracted from 14 individual adenocarcinomas and 2 specimens of normal lung tissue of untreated rats. In adenocarcinomas, elevated expression of IL-1 alpha (6/14), IL-1 beta (14/14), IL-3 (7/14), IL-4 (11/14), IL-5 (9/14), IL-6 (11/14) and IL-10 (8/14) was observed, compared with normal lung tissues. In contrast, no expression of IL-2 was detected in any case. The results suggest that preferential expression of these ILs and their complex networks may contribute to the development and progression of lung adenocarcinomas induced by BHP in rats.
  • W Kitayama; A Denda; E Okajima; T Tsujiuchi; Y Konishi
    CARCINOGENESIS 20 12 2305 - 2310 1999年12月 [査読有り]
     
    The anti-inflammatory drugs, aspirin and piroxicam, are known to possess chemopreventive potential against rat superficial urinary bladder carcinogenesis induced by N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN). Recently, we found similar inhibitory effects with a selective cyclooxygenase (COX)-2 inhibitor, nimesulide. In order to clarify the inhibitory mechanisms, we have further studied the expression of COX-2 protein in urinary bladder tumors induced by BBN in Fischer 344 male rats. For comparison, papillomatosis caused by uracil-induced urolithiasis, and normal epithelial cells, were also investigated. Western blot analysis revealed COX-2 protein to be barely expressed in the normal epithelial cells, whereas it was increased 13-22-fold in varying sizes of urinary bladder tumors and 7-fold in papillomatosis, Immunohistochemically, COX-2 protein was diffusely expressed in transitional cell carcinomas and nodulo-papillary hyperplasia but weakly expressed only in basal cells in simple hyperplasia and normal-looking surrounding epithelia, In papillomatosis, it was moderately expressed only in endothelial cells in stroma, These results indicate that COX-2 plays important roles in the development of preneoplastic and neoplastic lesions in the rat urinary bladder, and therefore could be a good target for chemoprevention of superficial lesions.
  • T Tsujiuchi; M Tsutsumi; Y Sasaki; M Takahama; Y Konishi
    JAPANESE JOURNAL OF CANCER RESEARCH 90 9 909 - 913 1999年09月 [査読有り]
     
    We have investigated aberrant methylation of CpG nucleotides (CpG sites) and gene expression of c-myc during hepatocarcinogenesis induced by a choline-deficient, L-amino acid-defined (CDAA) diet in rats. Male Fischer 344 rats, 6 weeks old, were continuously given a CDAA diet for 50 and 75 weeks and then killed, Macroscopically detectable nodules, which were histologically confirmed to be hyperplastic nodules (HNs) or well-differentiated hepatocellular carcinomas (HCCs), were dissected free from the surrounding tissue. Normal control liver was obtained from 6-week-old rats, Methylation of CpG sites of the c-myc gene was investigated in bisulfite-treated DNA isolated from normal liver, HNs and HCCs. All 33 cytosines in the 5'-upstream region of the c-myc gene were fully methylated in control liver and the 4 HNs. In contrast, these cytosines were completely unmethylated in 5 HCCs. Examination of the c-myc expression by reverse transcription-polymerase chain reaction (RT-PCR) analysis also showed a marked increase as compared to the low levels in normal livers and HNs, These results suggest that hypomethylation of the c-myc gene might play a critical role in malignant transformation from HN to HCC during CDAA diet-induced hepatocarcinogenesis in rats.
  • M Takahama; M Tsutsumi; T Tsujiuchi; K Nezu; K Kushibe; S Taniguchi; Y Kotake; Y Konishi
    CLINICAL CANCER RESEARCH 5 9 2506 - 2510 1999年09月 [査読有り]
     
    Expression of angiogenesis-associated genes was compared in 32 primary non-small cell lung carcinoma samples (14 adenocarcinomas, 17 squamous cell carcinomas, and 1 large cell carcinoma) and paired adjacent noncancerous lung tissues using a multiprobe RNase protection assay. Levels of Tie2, angiopoietin (Ang)-1, vascular endothelial growth factor (VEGF), and CD31 mRNAs were higher in cancers than in adjacent noncancerous tissues, in contrast to the fms-like tyrosine kinase (Flt)-1, Flt-4, Tiel, thrombin receptor, endoglin, and VEGF-C, for which no differences were evident. Overexpression did not seem to differ with histological type and pathological stage. Significant positive correlations were found between mRNA expression of Ang-l and those of Tie2 and CD31, and that of VEGF and those of Flt-1 and CD31, These findings suggest that Ang-1 and VEGF are important angiogenic factors in human non-small cell lung carcinomas.
  • M Yoshimoto; M Tsutsumi; K Iki; Y Sasaki; T Tsujiuchi; T Sugimura; K Wakabayashi; Y Konishi
    CANCER LETTERS 143 2 235 - 239 1999年09月 [査読有り]
     
    Effects of eight heterocyclic amines (HCAs) on pancreatic duct carcinogenesis were investigated in a rapid production model in hamsters. N-Nitrosobis(2-oxopropyl)amine (BOP) was given to effect initiation,followed by augmentation pressure consisting of four daily i.p. injections of 500 mg/kg DL-ethionine, a choline-deficient (CD) diet, a single i.p. injection of 800 mg/kg L-methionine and a s.c. injection of 20 mg/kg BOP. After two cycles of augmentation pressure, the HCAs 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), 3-amino-1-methyl-SH-pyrido[4,3-b]indole (Trp-P-2), 2-amino-9H-pyrido[2,3-b]indole (A alpha C), 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeA alpha C) or 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline [4,8-DiMeIQx) were administered in the diet for 50 or 70 days. The numbers of pancreatic ductal hyperplasias (H) and a total lesions including, atypical hyperplasia (AH), carcinomas in situ (CIS) and invasive carcinomas, were increased in hamsters given the diet containing 0.02% Trp-P-1 for 50 days. This result was confirmed and extended by the finding of increased numbers of invasive carcinomas in hamsters given 0.02% Trp-P-1 for 70 days. The number and incidence of invasive carcinomas were also elevated in hamsters given the diet containing 0.06% 4,8-DiMeIQx for 50 days. These results suggest a possible involvement of Trp-P-1 and 4,8-DiMeIQx in pancreatic duct carcinogenesis. (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.
  • T Tsujiuchi; M Tsutsumi; Y Sasaki; M Takahama; Y Konishi
    CANCER RESEARCH 59 16 3904 - 3907 1999年08月 [査読有り]
     
    To allow a study of beta-catenin mutations im hepatocellular carcinomas (HCCs) induced by exogenous and endogenous carcinogens, we induced tumors in male Fischer 344 rats with N-nitrosodiethylamine and a choline-deficient L-amino acid-defined diet, Administration of the former was followed by partial hepatectomy with colchicine to induce cell cycle disturbance and a selection pressure regimen (K. Ohashi et al., Cancer Res., 56: 3474-3479, 1996; M, Tsutsumi et al., Jpn. J. Cancer Res., 87: 5-9, 1996), HCCs were obtained after 42 weeks. With continuous choline-deficient L-amino acid-defined feeding, tumors were sampled after 75 weeks, Total RNA was extracted from individual lesions and mutations in the glycogen synthase kinase-3 beta phosphorylation consensus motif of beta-catenin mere investigated by reverse transcriptase-PCR-single-strand conformation polymorphism analysis followed by nucleotide sequencing, Changes were detected in 5 of 11 HCCs induced by the exogenous carcinogen. The observed shifts of C:G-->G:C or C:G-->A:T at codon 33 and G:C-->T:A transversions at codon 34 were associated with beta-catenin protein accumulation and confirmed by Western blot analysis. Only 2 of 15 HCCs induced in the endogenous carcinogenesis regimen demonstrated mutations, those being transitions of C:G-->T:A at codon 41 without amino acid alteration, These results suggest that different genetic pathways underlie exogenous and endogenous liver carcinogenesis in rats.
  • T Fukuda; A Kido; K Kajino; M Tsutsumi; Y Miyauchi; T Tsujiuchi; Y Konishi; O Hino
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 261 1 35 - 40 1999年07月 [査読有り]
     
    To identify differentially expressed genes between highly and low metastatic rat transplantable osteosarcomas, we applied a modified AFLP (amplified fragment length polymorphisms) method for cDNA subtraction. The specific point of our modification is selective amplification using suppression PCR technique after restriction enzyme cutting. Our cDNA-AFLP gave high reproducibility (about 95%) in mRNA patterns and enabled us to clone four dominantly expressed genes in a highly metastatic tumor line. Three showed homology with known genes, encoding Ki-67, a proliferation-associated effective marker of malignancy, type IV collagen alpha-3, a major component of basement membrane, and KIAA77 for which the function is unknown. Although one fragment showed no database homology, we revealed a derivation from the rat homologue of the Drosophila melanogaster diaphanous gene (Dia) by cloning of longer cDNA. Dia genes, known to affect actin filament formation, are downstream effecters of Rho small GTPase. The results suggest that alterations in the expression of cytoskeletal protein, basement membrane elements, and proliferative markers may be important for metastasis of osteosarcomas. (C) 1999 Academic Press.
  • A Kido; M Tsutsumi; K Iki; M Takahama; T Tsujiuchi; T Morishita; S Tamai; Y Konishi
    CANCER LETTERS 137 2 209 - 216 1999年04月 [査読有り]
     
    In the present experiment, the expression of matrix metalloproteinase (MMP)-2 and MMP-9, key proteins in the MMP family, and the tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2, antagonistic proteins against MMP-2 and MMP-9, respectively, were investigated by Northern blot analysis in rat transplantable osteosarcomas with high and low metastatic potencies. Two transplantable osteosarcomas, one induced with the carcinogen, 4-hydroxyaminoquinoline 1-oxide (4-HAQO) (COS, chemical carcinogen-induced osteosarcoma), and the other, a spontaneous lesion (SOS, spontaneous osteosarcoma), were repeatedly transplanted from lung nodules to generate lines with high metastatic potency, C-SLM (chemical carcinogen-induced osteosarcoma, selected lung metastatic lesions) and S-SLM (spontaneous osteosarcoma, selected lung metastatic lesions), respectively. MMP-9 was overexpressed in both S-SLM and C-SLM, and TIMP-2 in the case of S-SLM. Neither MMP-2 nor TIMP-1 was overexpressed in either of the transplantable osteosarcomas with high metastatic potentials. The active form MMP-9, studied by zymography, increased in S-SLM and C-SLM but not in SOS and COS. MMP-9 mRNA expression was highly correlated with the gelatinolytic activity of active form MMP-9 (r = 0.85, P < 0.0001) and with the activation ratio of MMP-9 (r = 0.83, P < 0.0001). However, the active form MMP-2 was not detectable in all cases. These results suggest that overexpression of MMP-9 mRNA is one of the essential factors in the acquisition of metastatic potential in rat transplantable osteosarcomas. (C) 1999 Published by Elsevier Science Ltd. All rights reserved.
  • M Takahama; M Tsutsumi; T Tsujiuchi; A Kido; H Sakitani; K Iki; S Taniguchi; S Kitamura; Y Konishi
    MOLECULAR CARCINOGENESIS 24 4 287 - 293 1999年04月 [査読有り]
     
    The expression of vascular endothelial growth factor (VEGF) and its receptors (VEGFRs), VEGFR-1/Flt-1 and VEGFR-2/Flk-1, was investigated by immunohistochemical and northern blot analysis during lung carcinogenesis by N-nitrosobis(2-hydroxypropyl)amine (BHP) in male Wistar rats. After BHP was given in the drinking water for 12 wk, the rats were maintained without further treatment until they were killed at 20-28 wk. Immunohistochemical studies revealed VEGF expression in almost all malignancies, the reaction being strongly positive in most adenocarcinomas (15 of 18; 83.3%) and squamous cell carcinomas (four of five; 80.0%), but less so in a total of 120 adenomas and 136 alveolar hyperplasias. In addition, VEGF mRNA and VEGFR mRNAs were found to be overexpressed in most adenocarcinomas and squamous cell carcinomas as well as in one to three of the five adenomas tested. The results indicated that VEGF and VEGFRs play important roles in the acquisition of malignant potential by preneoplastic lung lesions induced by BHP in rats. Moreover, overexpression of VEGF was related to upregulation of VEGFR-1/Flt-1 and VEGFR-2/Flk-1 expression in malignant and premalignant lung lesions. (C) 1999 Wiley-Liss, Inc.
  • A Kido; M Tsutsumi; K Iki; M Motoyama; M Takahama; T Tsujiuchi; T Morishita; K Tatsumi; S Tamai; Y Konishi
    JAPANESE JOURNAL OF CANCER RESEARCH 90 3 333 - 341 1999年03月 [査読有り]
     
    In the present experiment, we examined the effects of OPB-3206, 3S-[4-(N-hydroxyamino)-2R-isobutylsuccinyl]amino-1-methoxy-3,4-dihydrocarbostyril a novel metalloproteinase inhibitor, on the growth and metastasis of transplantable osteosarcomas (spontaneous osteosarcoma, selected lung metastatic lesions; S-SLM), which were previously established in rats. OPE-3206 inhibited the activities of interstitial collagenase, gelatinases A and B, and stromelysin in vitro. After oral administration to rats, its serum concentration peaked at 40 min and the drug was no longer detectable at 8 h. When OPE-3206 was orally administered at 0%, 0.1% and 0.4% in the diet for 4 weeks, starting 7 days after subcutaneous transplantation of osteosarcomas to male Fischer 344 rats, numbers of lung metastatic nodules were significantly reduced by the highest dose, while the growth of subcutaneous tumors was not affected. Zymographic analysis showed the presence of pro matrix metalloproteinase (proMMP)-2, proMMP-9 and MMP-9 activities in S-SLM. In animals fed 0.4% OPB-3206, the activity of proMMP-9 was increased, but that for MMP-9 had become undetectable. The results thus suggest that OPE-3206 selectively inhibits lung metastasis of rat transplantable osteosarcomas by inhibiting MMP-9 activation.
  • H Sakitani; M Tsutsumi; K Kadomatsu; S Ikematsu; M Takahama; K Iki; T Tsujiuchi; T Muramatsu; S Sakuma; T Sakaki; Y Konishi
    CARCINOGENESIS 20 3 465 - 469 1999年03月 [査読有り]
     
    The expression of midkine (MK) in lung tumors induced by N-nitrosobis(2-hydroxypropyl)amine (BHP) in rats was examined. The animals were administered 2000 p.p.m. of BHP in their drinking water for 12 weeks, then maintained without further treatment until being killed 20-28 weeks after the beginning of the experiment. MK mRNA expression of adenocarcinomas and squamous cell carcinomas assessed by means of the reverse transcriptase-polymerase chain reaction and northern blot analysis was significantly higher than in rat embryonic tissues (positive controls) and contrasted strongly with the lack in normal lungs. MK protein was detected immunohistochemically in 58.3% of alveolar hyperplasias, 92.3% of adenomas and 100% of adenocarcinomas and squamous cell carcinomas. The extent of staining significantly increased along with malignant progression in adenomatous (pre-)neoplastic lesions and tended to become more pronounced with malignant progression in squamous lesions. The results suggest that MK may play some essential roles in the development and progression of lung tumors induced by BHP in rats.
  • D Nakae; A Denda; Y Kobayashi; H Akai; H Kishida; T Tsujiuchi; Y Konishi; T Suzuki; M Muramatsu
    JAPANESE JOURNAL OF CANCER RESEARCH 89 11 1118 - 1125 1998年11月 [査読有り]
     
    Hepatocarcinogenesis initiated with N-nitrosodiethylamine (DEN) and that initiated by feeding of a choline-deficient, L-amino acid-defined (CDAA) diet mere compared in transgenic male Wistar rats harboring a rat glutathione S-transferase placental form (GST-P) gene (GST-P-Tg rats) and non-transgenic (N-Tg) rats. Fight-meek-old GST-P-Tg and N-Tg rats were administered DEN intraperitoneally at 100 mg/kg body weight, subjected to a selection procedure with 2-acetylaminofluorene and CCl4, and killed at the end of weeks 5 and 12. Other groups mere fed the CDAA diet for 12 weeks and killed. Five weeks after the DEN treatment, numbers and sizes of gamma-glutamyltransferase (GGT)- or GST-P-positive lesions and 8-hydroxyguanine (8-OHG) levels in the livers were significantly less in GST-P-Tg rats than in N-Tg rats. The lesion numbers were unchanged between the ends of weeks 5 and 12 in GST-P-Tg rats, but decreased in N-Tg rats. The lesion sizes were increased in GST-P-Tg rats, but unchanged in N-Tg rats. While the proliferating cell nuclear antigen labeling indices (PCNA L.I.) in and surrounding the lesions were decreased, more prominently in GST-P-Tg rats than in N-Tg rats, the 8-OHG levels mere also decreased but similarly in both cases. After 12 weeks on the CDAA diet, the lesion incidences, numbers and sizes, 8-OHG levels, PCNA L.I. in and surrounding the lesions, and liver injury were significantly less in GST-P-Tg rats than in N-Tg rats. These results indicate that insertion of a rat GST-P transgene alters the early phase of exogenous and endogenous rat hepatocarcinogenesis, presumably due to enhanced detoxification by GST-P expressed both transiently during the initiation and chronically in the altered hepatocyte populations.
  • D Nakae; Y Kotake; H Kishida; KL Hensley; A Denda; Y Kobayashi; W Kitayama; T Tsujiuchi; H Sang; CA Stewart; T Tabatabaie; RA Floyd; Y Konishi
    CANCER RESEARCH 58 20 4548 - 4551 1998年10月 [査読有り]
     
    Male Wister rats were fed a choline-deficient, L-amino acid-defined (CDAA) diet alone or in combination with a nitrone-based free radical trapping agent, phenyl N-tert-butyl nitrone (PBN) in the drinking water at the concentrations of 0.013, 0.065, and 0.130% for 12 weeks. PBN inhibited the changes that are normally induced in the livers of rats by the CDAA diet feeding, i.e., development of putative preneoplastic lesions, proliferation of connective tissue, reduction of glutathione S-transferase activity, formation of 8-hydroxyguanine in DNA and an increase in inducible cyclo-oxygenase (COX2) activity. PBN, however, did not prevent the increases in the COX2 mRNA or protein le,els brought on by the CDAA diet. These results indicate that the loss of glutathione S-transferase activity and COX2 induction may play significant roles in rat liver carcinogenesis by the CDAA diet and that PBN prevents neoplasia not only by its radical scavenging activity but also by inhibiting COX2 activity at the catalytic level.
  • A Kido; T Tsujiuchi; T Morishita; M Tsutsumi; M Takahama; Y Miyauchi; Y Mii; S Tamai; Y Konishi
    JAPANESE JOURNAL OF CANCER RESEARCH 89 10 1074 - 1081 1998年10月 [査読有り]
     
    To determine the role of telomerase activity in the growth of tumors in rats undergoing chemotherapy a comparison of the volumes of telomerase-positive transplantable osteosarcomas was made in rats treated with the antineoplastic agent cis-diammine dichloroplatinum (CDDP) or the angiogenesis inhibitor O-(chloroacetylcarbamoyl)fumagillol (AGM-1470). Male F344 rats, 8 weeks old, received transplants of macroscopic lung metastatic nodules into the subcutaneous hack space and treatment was started on day 14 thereafter CDDP was injected i.v. at doses of 0, 0.625, 1.25 and 2.5 mg/kg body weight (b.w.) and AGM-1470 was administered at total doses of 0, 2.5, 5 and 10 mg/kg b.w. over 2 weeks by osmotic pumps, also implanted into the subcutaneous back space, but remote from the transplanted tumors. On day 28, ail animals were killed for measurement of transplanted tumor size and determination of telomerase activities by telomeric repeat amplification protocol (TRAP) assay. The results showed telomerase activity to be highly correlated with the treated/non-treated (T/C) tumor size ratio (r = 0.96, P<0.0001). In a second experiment, CDDP at 2.5 mg/kg b.w and AGM-1470 at 10 mg/kg b.w., these being the most effective doses, were given as in the first experiment, and animals were serially killed on days 14, 21, 28, 35 and 42. Tumors in rats treated with CDDP and AGM-1470 showed 18.2% and 20.5% of the control telomerase activity on days 35 and 21, respectively, when tumor growth was inhibited. However, on day 42, the activities increased to 46.5% and 92.5%, this correlating with re-growth (r = 0.73, P<0.0001). These results suggest that decline of telomerase activity may be involved in tumor growth retardation induced by chemotherapeutic agents. This possibility clearly warrants further mechanistic studies.
  • Y Kobayashi; D Nakae; H Akai; H Kishida; E Okajima; W Kitayama; A Denda; T Tsujiuchi; A Murakami; K Koshimizu; H Ohigashi; Y Konishi
    CARCINOGENESIS 19 10 1809 - 1814 1998年10月 [査読有り]
     
    The effects of 1'-acetoxychavicol acetate (ACA) on endogenous rat liver carcinogenesis because of chronic feeding of a choline-deficient, L-amino acid-defined (CDAA) diet were examined. Male Fischer 344 rats, 6 weeks old, received the CDAA diet containing ACA at doses of 0, 0,005, 0.010 and 0.050% for 12 weeks and were then killed. ACA decreased the numbers of putative preneoplastic, glutathione S-transferase placental form (GST-P)-positive, focal lesions developing in the livers of rats fed the CDAA diet but did not alter their sizes. At the same time, ACA reduced the levels of 8-hydroxyguanine, a parameter of oxidative DNA damage, but did not significantly affect generation of 2-thiobarbituric acid-reacting substances, indicators of oxidative extra-DNA damage, or hepatocyte proliferation. Furthermore, ACA did not exert any significant effects on the numbers or sizes of GST-P-positive lesions in the livers of fats when administered between weeks 2 and 8 after initiation with a single i.p. dose of 200 mg/kg body wt of N-nitrosodiethylamine. These results indicate that ACA prevents the CDAA diet-associated induction of putative preneoplastic lesions by reduction of oxidative DNA damage but does not affect their subsequent growth.
  • K Iki; T Tsujiuchi; T Majima; H Sakitani; M Tsutsumi; M Takahama; M Yoshimoto; D Nakae; T Tsunoda; Y Konishi
    CANCER LETTERS 131 2 185 - 190 1998年09月 [査読有り]
     
    Telomerase activities in intrahepatic cholangiocarcinomas induced by N-nitrosobis (2-oxopropyl)amine (BOP) in female hamsters were determined using a telomeric repeat amplification protocol (TRAP) assay followed by densitometric quantification. Each determination was repeated to confirm the results and telomerase activity was also detected by gel electrophoresis. An increase was evident in all of 10 cholangiocarcinomas examined, with levels ranging from 2.48 to 4.40 times the normal liver value by densitometric quantification. This finding of a consistent increase suggests that telomerase activation is involved in the development of intrahepatic cholangiocarcinomas and immortalization of cancer cells. (C) 1998 Elsevier Science Ireland Ltd. All rights reserved.
  • T Tsujiuchi; M Tsutsumi; Y Konishi
    EXPERIMENTAL AND TOXICOLOGIC PATHOLOGY 50 4-6 371 - 374 1998年09月 [査読有り]
  • A Denda; T Endoh; Q Tang; T Tsujiuchi; D Nakae; Y Konishi
    MUTATION RESEARCH-FUNDAMENTAL AND MOLECULAR MECHANISMS OF MUTAGENESIS 402 1-2 279 - 288 1998年06月 [査読有り]
     
    Effects of inhibitors of arachidonic acid (AA) cascade on the development of fatty liver, cirrhosis, glutathione S-transferase placental form (GST-P)-positive preneoplastic nodules, neoplastic nodules and generation of 8-hydroxydeoxyguanosine (8-OHdG), caused by a choline-deficient, L-amino acid-defined (CDAA) diet, were examined in Fischer 344 male rats by feeding CDAA diet supplemented with the inhibitors for 12 and 30 weeks. None of the inhibitors affected fatty liver. Among cyclooxygenase (COX) inhibitors, an irreversibly acting acetylsalicylic acid and a long-acting piroxicam, and to a much lesser extent the short-acting ibuprofen but not indomethacin, inhibited the development of cirrhosis, GST-P-positive and neoplastic nodules and generation of 8-OHdG. A phospholipase A(2) inhibitor p-bromophenacylbromide (BPB) also exerted similar but lesser extent of inhibitory effects. Lipoxygenase inhibitors quercetin and nordihydroguiaretic acid inhibited GST-P-positive nodules but not cirrhosis or 8-OHdG. Present results suggest that perturbed AA cascade, particularly augmented COX pathway, might play key roles in the causation of liver lesions in the CDAA diet model. (C) 1998 Elsevier Science B.V. All rights reserved.
  • K Honoki; T Mori; M Tsutsumi; T Tsujiuchi; A Kido; T Morishita; Y Miyauchi; Y Dohi; Y Mii; S Tamai; Y Konishi
    CANCER LETTERS 127 1-2 221 - 228 1998年05月 [査読有り]
     
    We have established three cloned cell lines (COS1NR, COS2NR and COS4NR) from the lung metastatic nodule of a highly metastatic variant of rat transplantable osteosarcoma, C-SLM. All three clones shared the same morphological characteristics and tumorigenicity, but their growth rates in vitro and metastatic ability in vivo differed from each other. Single-strand conformation polymorphism (SSCP) analysis revealed all three clones to have the same p53 gene mutation and parent C-SLM tumor. On the other hand, Northern blot analysis showed a different pattern of expression for the genes, c-fos, c-jun, c-Ha-ras, transin (rat stromelysin), bone Gla protein (osteocalsin) and nm23/NDP kinase. These results indicate the presence of a heterogeneous cell population in terms of the different pattern of gene expression in a lung metastatic nodule of rat osteosarcoma and the present newly established cell lines will be useful for further investigation of the biological behavior of osteosarcomas. (C) 1998 Elsevier Science Ireland Ltd.
  • H Maruyama; S Sugihara; K Ishihara; K Sada; M Tsutsumi; T Tsujiuchi; D Nakae; Y Konishi
    PATHOLOGY INTERNATIONAL 48 5 403 - 411 1998年05月 [査読有り]
     
    An autopsy case of systemic mast cell disease (SMCD) without primary skin lesions in a 57-year-old Japanese male is described. Initially the patient was suspected of having liver cirrhosis or malignant lymphoma because of hepatomegaly and lymph node enlargement on admission. However, a lymph node biopsy and bone marrow aspiration conducted on his third admission indicated a SMCD because of the existence of metachromatic cell aggregates stained with toluidine blue. At autopsy, the diagnosis was confirmed because the proliferating cells were histochemically proven to be mast cells by naphthol AS.D chloroacetate esterase, Giemsa and alcian blue, in addition to toluidine blue staining. The intra-abdominal and retroperitoneal lymph nodes were replaced by mast cell aggregates, which caused the splenic infarction and bilateral hydronephrosis, with infiltration of mast cells into the spleen and kidneys also being apparent. Mast cell infiltration was similarly found in the bone marrow, liver, ileum and ascending colon. Immunohistochemically, the mast cells were positive for antibodies of alpha(1)-antichymotrypsin, CD45 (LCA), CD43 (MT-1), CD45R (MB-1) and the oncoprotein c-kit, Electron microscopic examination using formalin-fixed tissue gave supportive evidence of a mast cell origin for the lesions.
  • Y Konishi; M Tsutsumi; T Tsujiuchi
    PANCREAS 16 3 300 - 306 1998年04月 [査読有り]
     
    In this article, we introduce our rapid-production model for pancreatic duct adenocarcinomas and describe the mechanisms of pancreatic duct carcinogenesis so far elucidated in Syrian golden hamsters. It is evident that a series of histogenetic steps are involved, leading from hyperplasia through atypical hyperplasia to intraductal carcinoma and invasive carcinoma. As DNA alters, K-ras mutation appears to be an early event, whereas p53 mutations generally occur in the tumor-progression phase. The induced cancer cells may show autocrine growth, secreting TGF-alpha and vascular endothelial growth factor (VEGF), and are immortalized with a shortened TRF length and increased telomerase activity. The rapid-production model of pancreatic duct adenocarcinomas has not only provided a major stimulus to understanding induction mechanisms but should also serve as a bioassay to facilitate the identification of dietary risk factors and the search for appropriate chemopreventive or chemotherapeutic agents or both to help control this deadly disease.
  • H Sakitani; T Tsujiuchi; K Kobitsu; A Kido; K Iki; M Takahama; M Nakamura; T Sakaki; D Nakae; Y Konishi; M Tsutsumi
    CANCER LETTERS 126 2 157 - 164 1998年04月 [査読有り]
     
    The question of whether the changes in telomerase activity and/or the alteration of the p53 gene are involved in the development of oligo-astrocytomas induced by N-ethyl-N-nitrosourea (ENU) in rats was addressed. Telomerase activity levels of oligo-astrocytomas, including early neoplastic lesions, were significantly increased as compared to the normal controls, correlating with the degree of malignancy. In contrast, no mutations of p53 exons 5-7 were found in early neoplastic lesions or oligo-astrocytomas. These results indicate that the activation of telomerase occurs during astrocytoma carcinogenesis and contributes to the development of brain tumors, but the alterations of p53, at least on exons 5-7, may not be involved in this process. (C) 1998 Elsevier Science Ireland Ltd.
  • M Takahama; M Tsutsumi; T Tsujiuchi; A Kido; E Okajima; K Nezu; T Tojo; K Kushibe; S Kitamura; Y Konishi
    JAPANESE JOURNAL OF CLINICAL ONCOLOGY 28 3 176 - 181 1998年03月 [査読有り]
     
    Background: Angiogenesis is an essential factor for progression and metastases in solid tumors. It has been reported that several angiogenic factors play a role in the regulation of angiogenesis. Vascular endothelial growth factor (VEGF) is one of the most important molecules in angiogenesis, We investigated expressions of VEGF in a series of lung carcinomas with regard to clinicopathological factors. Method: VEGF expression was investigated by use of immunohistochemical studies and Northern blot analysis, using 155 primary and 26 metastatic lung carcinomas for the immunohistochemical studies and 10 primary and two metastatic lung carcinomas for the Northern blot analysis. All lesions were resected at surgery. Results: The frequencies for positive VEGF expression were 64 of 74 (86.5%) adenocarcinomas, 38 of 67 (56.7%) squamous cell carcinomas, four of four (100%) large cell carcinomas, two of three (66.7%) adenosquamous carcinomas and one of five (20%) small-cell carcinomas, the degree of positivity generally being greater in well differentiated tumors, The majority of metastatic foci from adenocarcinomas and squamous cell carcinomas at other sites were also positive (76.5 and 66.7%, respectively). VEGF expression did not correlate with clinicopathological factors such as tumor size or pathological stage, but pathological stage adenocarcinoma cases positive for VEGF demonstrated a shorter disease-free period when followed up for 48 months than those cases expressing VEGF negatively. Conclusions: The results indicated that VEGF expression was frequently detected in non-small-cell lung cancers and suggested that VEGF might relate to the disease-free period of the patients with early adenocarcinomas.
  • T Tsujiuchi; M Tsutsumi; A Kido; M Takahama; H Sakitani; K Iki; Y Sasaki; A Denda; Y Konishi
    CANCER LETTERS 122 1-2 115 - 120 1998年01月 [査読有り]
     
    The regulation of telomerase activity during regeneration induced by two-thirds partial hepatectomy (PH) was investigated in 6-week-old male F344 rats. Groups of animals were serially sacrificed 0, 6, 16, 24, 36 and 72 h after the operation and telomerase activity was determined by the telomeric repeat amplification protocol (TRAP) assay followed by densitometric quantification. DNA synthesis was immunohistochemically quantified in terms of bromodeoxyuridine (BrdU) incorporation. The expression levels of telomerase RNA were examined by Northern blot analysis. Telomerase activity was increased significantly from 6 to 36 h but had decreased to close to the normal levels after 72 h. DNA synthesis reached a maximum 24 h after PH. However, the expression levels of telomerase RNA did not change during regeneration. The results suggest that telomerase is actively regulated by unknown mechanisms throughout the cell cycle in regenerating rat hepatocytes. (C) 1998 Elsevier Science Ireland Ltd.
  • K Tamura; D Nakae; K Horiguchi; H Akai; Y Kobayashi; N Andoh; H Satoh; A Denda; T Tsujiuchi; H Yoshiji; Y Konishi
    CARCINOGENESIS 18 11 2133 - 2141 1997年11月 [査読有り]
     
    The effects of N-(4-hydroxyphenyl)retinamide (4-HPR) and all-trans-retinoic acid (tRA) on the exogenous and endogenous models of rat liver carcinogenesis respectively using diethylnitrosamine (DEN) and a choline-deficient, L-amino acid-defined (CDAA) diet were studied, For the exogenous study, male Fischer 344 rats, 6 weeks old, were given a single i.p. dose of 200 mg/kg body wt of DEN, partially hepatectomized at week 3, administered 4-HPR at doses of 0, 0.04, 0.08 and 0.16% or tRA at 0, 0.004, 0.008 and 0.015% in diet from week 2 for 6 weeks, and killed at the end of week 8, For the endogenous study, rats were fed the CDAA diet containing 4-HPR or tRA for 12 weeks and killed at the end of week 12, 4-HPR decreased the numbers and sizes of the glutathione S-transferase placental form-positive foci, assayed as putative preneoplastic lesions, the levels of 8-hydroxyguanine (8-OHG), a parameter of oxidative DNA damage, and the bromodeoxyuridine labeling indices (BrdU L.I.) by all three doses in the DEN-initiated case and, more prominently, in the CDAA diet-associated case, In contrast, while tRA failed to exert inhibitory effects apparently on foci development, 8-OHG formation or BrdU labeling in the DEN-initiated case, it reduced the numbers and sizes of the foci, the 8-OHG levels and the BrdU L.I. by all three doses in the CDAA diet-associated case, Furthermore, both 4-HPR and tRA inhibited the CDAA diet-associated induction of hepatocyte necrosis and connective tissue increase but not intrahepato-cellular fat accumulation, These results indicate that 4-HPR exerts chemopreventive effects against the exogenous and endogenous rat liver carcinogenesis, while tRA can inhibit only the latter.
  • M Tsutsumi; T Tsujiuchi; O Ishikawa; T Majima; M Yoshimoto; Y Sasaki; T Fukuda; H Oohigashi; Y Konishi
    JAPANESE JOURNAL OF CANCER RESEARCH 88 10 971 - 976 1997年10月 [査読有り]
     
    Telomerase is a key enzyme with regard to immortalization of cancer cells and increased activity has been demonstrated in various human malignant neoplasms. Since little is known of its role in pancreatic cancers, we investigated changes in telomerase activity in human pancreatic duct adenocarcinomas and compared the frequency of increased telomerase activity with the presence of K-ras gene mutations. The samples were obtained from 38 pancreatic duct adenocarcinomas and 7 tumor surrounding tissues at surgical resection. Telomerase activity was examined by telomeric repeat amplification protocol assay and terminal restriction fragment (TRF) length was examined by Southern analysis. K-ras mutation was examined by means of polymerase chain reaction-single strand conformation polymorphism analysis. Among 38 pancreatic carcinomas, 32 (84%) exhibited increased telomerase activities with no apparent relation to the histological type of tumor, tumor size, regional lymphnode involvement and distant metastasis or clinical stage. In tissue surrounding the tumor, telomerase activity was not detected. TRF length tended to be reduced in pancreatic carcinomas. Mutations of K-ras gene were found in 24 out of the 38 (63%) cases. Among the 38 cases, 14 showed increased telomerase activity without K-ras mutation and 4 cases showed K-ras mutation without telomerase activity. These results suggest that increased telomerase activity might be a sensitive genetic diagnostic marker and could be a target for future therapy of pancreatic duct carcinomas.
  • A Denda; T Endoh; W Kitayama; Q Tang; O Noguchi; Y Kobayashi; H Akai; E Okajima; T Tsujiuchi; M Tsutsumi; D Nakae; Y Konishi
    CARCINOGENESIS 18 10 1921 - 1930 1997年10月 [査読有り]
     
    Previously, we have reported that aspirin, a cyclooxygenase (COX) inhibitor, can prevent the fibrosis, cirrhosis and generation of oxidative DNA damage, and the associated development of glutathione-S-transferase placental form (GST-P)-positive preneoplastic liver nodules, caused by a choline-deficient, L-amino acid-defined (CDAA) diet in rats. In the present study, in order to elucidate the role of COX pathway in liver lesion-induction by a CDAA diet, the modulatory effects of other distinct chemical classes of COX inhibitors were examined. A long-acting example, piroxicam (PIRO) (at doses of 0.01, 0.02, 0.04 and 0.06%) and the short-acting ibuprofen (IBU) (at doses of 0.02, 0.04 and 0.06%) and indomethacin (IND) (at doses of 0.005 and 0.008%) were administered in the CDAA diet to male F344 rats, and animals were killed after 12 and 30 weeks. In another experiment, IND was given in drinking water at doses of 0.001, 0.002 and 0.004%. None of the inhibitors affected the development of fatty liver caused by a CDAA diet, but PIRO at doses higher than 0.04%, strongly inhibited the development of GST-P-positive and neoplastic nodules as well as fibrosis, cirrhosis and formation of 8-hydroxydeoxyguanosine (8-OHdG) adducts. IBU at the highest dose also exhibited similar but much less pronounced inhibitory effects. With IND, there was only a tendency for inhibition with no clear dose-dependence. The results together with our previous findings, indicate that relatively strong COX inhibitors, acting irreversibly like aspirin or for extended periods like PIRO, can prevent the endogenous hepatocarcinogenesis associated with a CDAA diet, although not the development of a fatty liver, suggesting that an augmented COX pathway might play key roles in the causation of liver lesions in this model.
  • T Majima; T Tsujiuchi; M Tsutsumi; T Tsunoda; Y Konishi
    CANCER LETTERS 118 1 47 - 53 1997年09月 [査読有り]
     
    An experimental model for the induction of extrahepatic biliary duct carcinomas in hamsters given cholecystoduodenostomy with dissection of the extrahepatic duct at the distal end of the common duct (CDDB) followed by N-nitrosobis(2-oxopropyl)amine (BOP) has been reported [Tajima et al. (1994) Jpn, J. Cancer Res., 85, 780-788]. The CDDB procedure greatly accelerates cell turnover in the biliary epithelium. In the present experiment, mutations of K-ras and p53 genes in the induced lesions were investigated by the reverse transcriptase-polymerase chain reaction-single strand conformation polymorphism (RT-PCR-SSCP) method followed by direct sequencing. Mutations of K-ras, involving a G to A transition in second position of codon 12 of K-ras exon I, were detected in six out of eight (75%) extrahepatic bile duct carcinomas and six out of eleven (54.5%) pancreatic duct carcinomas. However, no mutations of p53 were observed in either tumor type. The results indicate an association between anomalous pancreaticobiliary junction and development of biliary carcinomas that may be pertinent to the human situation, and indicate that conditions of the model predispose to mutations occurring in K-ras but not p53. (C) 1997 Elsevier Science Ireland Ltd.
  • T Tsujiuchi; L Yeleswarapu; Y Konishi; B Lombardi
    BRITISH JOURNAL OF CANCER 76 1 15 - 20 1997年07月 [査読有り]
     
    Male F-344 rats were fed for 15 weeks a methyl-deficient L-amino acid defined diet containing 0.05% DL-ethionine. Nodules protruding from the surface of the liver were dissected free of surrounding tissue, and polyadenylated RNA isolated from the nodules was reverse transcribed. The region of the p53 gene comprising codons 120-290 was amplified by the polymerase chain reaction, and cDNAs were sequenced. Mutations were detected in nodules obtained from 7 of 12 rats. In all seven cases, the same two point mutations were present. The first was at the first base of codon 246 and consisted of a C-->T transition (C:G-->T:A, Arg-->Cys), while the second was at the second base of codon 247 and consisted of a G-->T transversion (G:C-->T:A, Arg-->Leu). It is concluded that the hepatocarcinogen ethionine induces specific hot-spot p53 gene mutations; this is in contrast to the mutations at Various sites previously observed to occur in rats fed a hepatocarcinogenic methyl-deficient diet alone. The results also provide the first evidence that ethionine is mutagenic in the rat.
  • A Kido; T Tsujiuchi; M Tsutsumi; M Takahama; Y Miyauchi; Y Mii; S Tamai; Y Konishi
    CANCER LETTERS 117 1 67 - 71 1997年07月 [査読有り]
     
    Previously, we reported the establishment of two transplantable osteosarcomas in rats, one induced by local application of a carcinogen, 4-hydroxyamino quinoline 1-oxide (4-HAQO), and another which developed spontaneously, and their subdivision into four lines with high and low metastatic potential to the lung. In the present study, activation of telomerase was investigated by the telomeric repeat amplification protocol (TRAP) assay followed by densitometric quantification. Telomerase activity was found to be elevated in all four lines without any link to the metastatic potential. Thus the spontaneous osteosarcoma (SOS) and derived metastatic lesions (S-SLM) demonstrated a 20.1-23.5-fold increase and the chemical carcinogen (4HAQO)-induced osteosarcoma (COS) and metastatic lesions (C-SLM) were 18.4-19.1-fold elevated as compared to the value for abdominal muscle. The results suggest that activation of telomerase occurs in rat osteosarcomas but that it is not directly involved in determining their metastatic potential. (C) 1997 Elsevier Science Ireland Ltd.
  • K Tamura; D Nakae; K Horiguchi; H Akai; Y Kobayashi; H Satoh; T Tsujiuchi; A Denda; Y Konishi
    JAPANESE JOURNAL OF CANCER RESEARCH 88 4 356 - 362 1997年04月 [査読有り]
     
    The effects of green tea extract (GTE) on exogenous and endogenous models of rat liver carcinogenesis using diethylnitrosamine (DEN) and a choline-deficient, L-amino acid-defined (CDAA) diet were studied. For the exogenous carcinogenesis study, male Fischer 344 rats, 6 weeks old, were given a single intraperitoneal dose of 200 mg/kg body weight of DEN, partially hepatectomized at week 3, and administered GTE at doses of 0, 0.01 and 0.1% in the drinking water from week 2 for 10 weeks. For the endogenous carcinogenesis study, rats were fed the CDAA diet and simultaneously given GTE for 12 weeks. All rats were killed at the end of week 12. After DEN-initiation, the apparent numbers of glutathione S-transferase placental form-positive foci, assayed as putative preneoplastic lesions, were decreased by the administration of GTE, though their sizes were not altered. In contrast, GTE did not significantly reduce the numbers of the lesions induced by the CDAA diet or affect their sizes. While the levels of 8-hydroxyguanine, a parameter of oxidative DNA damage, were reduced by the GTE administration in both experimental models, GTE did not protect against the CDAA-diet-associated liver tissue damage in terms of either histology or plasma marker enzyme levels. We conclude that, while GTE may be a possible chemopreventive agent for nitrosamine-initiated hepatocarcinogenesis in the absence of chronic hepatocyte damage, it does not significantly inhibit lesion development in hepatocarcinogenesis associated with the CDAA diet, a cirrhosis-associated model.
  • Y Murata; A Denda; H Maruyama; D Nakae; M Tsutsumi; T Tsujiuchi; Y Konishi
    FUNDAMENTAL AND APPLIED TOXICOLOGY 36 1 90 - 93 1997年03月 [査読有り]
     
    The toxicity and carcinogenic potential of 2-methylnaphthalene (2-MN) were examined in B6C3F(1) mice. Groups of 50 male and 50 female mice were given diets containing 0, 0.075, and 0.15% 2-MN for 81 weeks. Both 0.075 and 0.15% 2-MN caused pulmonary alveolar proteinosis at high incidence: 55.1 and 45.8% in females and 42.9 and 46.9% in males, respectively, The incidences of total lung tumors, including bronchiolar/alveolar adenomas and carcinomas, were 20.4 and 12.2% in male mice given 0.075 and 0.15% 2-MN, respectively, the former value being significantly increased compared with the 4.1% in control males. However, in the respective incidences of the adenomas and carcinomas, neither intergroup differences nor dose dependencies were observed. The incidences of other tumors did not differ between mice treated with 2-MN and the controls. The results indicated that 2-MN induces pulmonary alveolar proteinosis but does not possess unequivocal carcinogenic potential in B6C3F, mice. (C) 1997 Society of Toxicoloty.
  • K Kobitsu; M Tsutsumi; T Tsujiuchi; F Suzuki; A Kido; E Okajima; T Fukuda; T Sakaki; Y Konishi
    MOLECULAR CARCINOGENESIS 18 3 153 - 159 1997年03月 [査読有り]
     
    Recently, shortened telomere length and increased telomerase activity have been demonstrated in various human cancers. In the study reported here, we ascertained whether gene changes are characteristic of pancreatic cancers. Hamster duct carcinomas and cell lines were investigated by Southern blot analysis for telomere restriction fragment (TRF) length and by the telomeric repeat amplification protocol (TRAP) assay for telomerase activity. Comparison with normal pancreas and spleen revealed shortened TRF length and markedly increased telomerase activity in primary pancreatic duct carcinomas induced by the rapid-production model as well as in a transplantable carcinoma a nd the cell lines. The enzyme level was 86.0-215.7 times the low levels found in control pancreas and spleen tissues. Late-passage Syrian hamster embryo cells, known to be immortalized and tumorigenic, had shorter TRFs than the original cells in primary culture did. These results indicate that hamster pancreatic duct carcinoma cells are immortalized, with the potential for proliferation ad infinitum, and provide a model for basic therapeutic research into the substances targeting telomerase. (C) 1997 Wiley-Liss, Inc.
  • A Kido; T Tsujiuchi; M Tsutsumi; M Takahama; E Okajima; K Kobitsu; Y Miyauchi; Y Mii; S Tamai; Y Konishi
    CANCER LETTERS 112 1 5 - 10 1997年01月 [査読有り]
     
    Previously, we reported the establishment of two transplantable osteosarcomas, one induced by local application of a carcinogen, 4-hydroxyamino quinoline 1-oxide(4-HAQO), and another which developed spontaneously in rats, and their subdivision into four lines with high and low metastatic potential to the lung. In the present study, mutations of p53 and Ki-ras genes were investigated by PCR and SSCP followed by direct sequencing, and the amplification of the mdm2 gene was assessed by Southern blot analysis. Mutations of p53 in exon 7 were detected in 4-HAQO-induced transplantable osteosarcomas, but not their spontaneous counterparts, irrespective of the metastatic potentials. Direct sequencing revealed a CGC to CAC transition with an amino acid change of Arg to His, at codon 246. Neither Ki-ras mutations nor mdm2 amplification were detected in airy of the transplantable tumors. The results suggest that while p53 mutations occurred during osteosarcoma development by 4-HAQO without mdm2 amplification and Ki-ras mutation does not contribute to osteosarcoma development in rats. (C) 1997 Elsevier Science Ireland Ltd.
  • T Tsujiuchi; M Tsutsumi; A Kido; K Kobitsu; M Takahama; T Majima; A Denda; D Nakae; Y Konishi
    JAPANESE JOURNAL OF CANCER RESEARCH 87 11 1111 - 1115 1996年11月 [査読有り]
     
    Activation of telomerase has been reported in several human cancers, including hepatocellular carcinomas (HCCs). We investigated telomerase activity during hepatocarcinogenesis induced by a choline-deficient L-amino acid-defined (CDAA) diet in rats. Male F344 rats were given a CDAA diet or a choline-supplemented L-amino acid-defined (CSAA) diet from 6 weeks of age for 75 weeks, and subgroups were killed 10 weeks, 50 weeks and 75 weeks after the beginning of the experiment. Hyperplastic nodules and HCCs were noted in rats fed a CDAA diet for 50 weeks and 75 weeks, respectively. Normal control liver specimens were obtained from 6-week-old rats. Telomerase activity was assessed by using a telomeric repeat amplification protocol (TRAP). Normal liver and background parenchyma of rats fed either of the diets for 10 weeks or 50 weeks showed weak telomerase activity. In contrast, markedly increased levels were demonstrated in hyperplastic nodules and HCCs. These results suggest that increased telomerase activity may be a biological feature of preneoplastic lesions that evolve to HCCs in rat liver.
  • T Tsujiuchi; A Kido; D Nakae; M Takahama; T Majima; K Kobitsu; E Okajima; M Tsutsumi; A Denda; Y Konishi
    CANCER LETTERS 108 1 137 - 141 1996年11月 [査読有り]
     
    Mutations of Ki-ras and p53 genes in hepatocellular carcinomas (HCCs) induced by the choline deficient L-amino acid defined (CDAA) diet in rats were investigated by polymerase chain reaction (PCR), single strand conformation polymorphism (SSCP) analysis followed by direct sequencing. Male Fischer 344 rats, 6 weeks old, were continuously given a CDAA diet for 70 weeks and then sacrificed. Macroscopically detectable nodules which were histologically confirmed to be well-differentiated HCCs were dissected free from the surrounding tissue and subjected to gene mutation analysis along with samples of non-tumor areas. Conformational change in the Ki-ras gene was detected in 1 out of 7 HCCs, involving a GGC to GTC transversion at codon 13. No p53 mutations were detected in 7 HCCs and also neither Ki-ras nor p53 mutations were found in non-tumor areas. The results suggest that neither Ki-ras nor p53 genes play an important role in hepatocarcinogenesis caused by long term expose to a CDAA diet in rats.
  • T Tsujiuchi; A Kido; D Nakae; M Takahama; T Majima; K Kobitsu; E Okajima; M Tsutsumi; A Denda; Y Konishi
    CANCER LETTERS 108 1 137 - 141 1996年11月 
    Mutations of Ki-ras and p53 genes in hepatocellular carcinomas (HCCs) induced by the choline deficient L-amino acid defined (CDAA) diet in rats were investigated by polymerase chain reaction (PCR), single strand conformation polymorphism (SSCP) analysis followed by direct sequencing. Male Fischer 344 rats, 6 weeks old, were continuously given a CDAA diet for 70 weeks and then sacrificed. Macroscopically detectable nodules which were histologically confirmed to be well-differentiated HCCs were dissected free from the surrounding tissue and subjected to gene mutation analysis along with samples of non-tumor areas. Conformational change in the Ki-ras gene was detected in 1 out of 7 HCCs, involving a GGC to GTC transversion at codon 13. No p53 mutations were detected in 7 HCCs and also neither Ki-ras nor p53 mutations were found in non-tumor areas. The results suggest that neither Ki-ras nor p53 genes play an important role in hepatocarcinogenesis caused by long term expose to a CDAA diet in rats.
  • T Tsujiuchi; M Tsutsumi; A Kido; K Kobitsu; M Takahama; T Majima; A Denda; D Nakae; Y Konishi
    JAPANESE JOURNAL OF CANCER RESEARCH 87 11 1111 - 1115 1996年11月 
    Activation of telomerase has been reported in several human cancers, including hepatocellular carcinomas (HCCs). We investigated telomerase activity during hepatocarcinogenesis induced by a choline-deficient L-amino acid-defined (CDAA) diet in rats. Male F344 rats were given a CDAA diet or a choline-supplemented L-amino acid-defined (CSAA) diet from 6 weeks of age for 75 weeks, and subgroups were killed 10 weeks, 50 weeks and 75 weeks after the beginning of the experiment. Hyperplastic nodules and HCCs were noted in rats fed a CDAA diet for 50 weeks and 75 weeks, respectively. Normal control liver specimens were obtained from 6-week-old rats. Telomerase activity was assessed by using a telomeric repeat amplification protocol (TRAP). Normal liver and background parenchyma of rats fed either of the diets for 10 weeks or 50 weeks showed weak telomerase activity. In contrast, markedly increased levels were demonstrated in hyperplastic nodules and HCCs. These results suggest that increased telomerase activity may be a biological feature of preneoplastic lesions that evolve to HCCs in rat liver.
  • K Ohashi; M Tsutsumi; T Tsujiuchi; K Kobitsu; E Okajima; Y Nakajima; H Nakano; M Takahashi; Y Mori; Y Konishi
    CANCER RESEARCH 56 15 3474 - 3479 1996年08月 [査読有り]
     
    The effects of a colchicine-induced M-phase block of regeneration after partial hepatectomy on early-stage liver carcinogenesis were studied in rats. When administered 1 or 3 days after N-diethylnitrosamine initiation and partial hepatectomy, colchicine increased the mitotic index of regenerating hepatocytes at days 4-6 without evidence of liver cell necrosis. When the protocol was combined with a selection procedure (E. Cayama et al., Nature (Lond.), 275: 60-62, 1978), a significant increase in the size but not number of gamma-glutamyltranspeptidase-positive foci at week 5 was observed in a colchicine dose-dependent manner. This was associated with an elevated incorporation of 5-bromo-2-deoxyuridine into the gamma-glutamyltranspeptidase-positive cells. In a longer-term experiment, the numbers, sizes, and 5-bromo-2-deoxyuridine labeling index of persistent nodules were increased significantly in colchicine-treated rats at week 9. This was associated with significant increases in the incidences and numbers of hepatocellular carcinomas at week 42. The above results raise the interesting possibility that a cell cycle disturbance in the early stage of liver carcinogenesis provides a persisting growth advantage for initiated cells, resulting in enhanced growth of foci and persistent nodules that evolve into hepatocellular carcinomas.
  • E Kobayashi; T Tsujiuchi; D Nakae; Y Mizumoto; N Andoh; T Endoh; H Kitada; M Tsutsumi; A Denda; Y Konishi
    EXPERIMENTAL AND TOXICOLOGIC PATHOLOGY 48 4 275 - 282 1996年06月 [査読有り]
     
    Effects of N,N'-diphenyl-p-phenylenediamine (DPPD), an antioxidant, on liver carcinogenesis caused by a choline-deficient L-amino acid-defined (CDAA) diet containing ethionine were studied in Fischer 344 rats. Male animals, 6 weeks old, were fed a CDAA diet, a choline-supplemented L-amino acid-defined (CSAA) diet or a CDAA diet containing 0.05% ethionine with or without 0.2% DPPD. Histological changes and lesions positive for gamma-glutamyltransferase (GGT) were analyzed 12 weeks after the beginning of the experiment. The levels of 8-hydroxyguanine (8-OHGua) in DNA and 2-thiobarbituric acid-reacting substances (TEARS) were measured as the parameters for cellular oxidative damage after 4 and 11 days of treatment. Expression of c-myc and c-Ha-vas was also investigated in relation to cell proliferation after 2, 4, 8 and 11 days. Histologically, development of diffuse fatty liver observed in rats fed a CDAA diet was inhibited, while massive oval cell proliferation and cholangiofibrosis resulted from the addition of ethionine with/without DPPD. The sizes but not numbers of GGT-positive lesions seen in the liver of rats fed a CDAA diet were increased and the levels of 8-OHGua formation and TEARS generation were also increased by the ethionine supplement. Both numbers and sizes of GGT-positive lesions were decreased and the level of TEARS, but not 8-OHGua, was decreased by adding DPPD. The increased expression of c-myc and c-Ha-ras detected in the liver of rats fed a CDAA diet was further increased by addition of ethionine and again reduced by DPPD. These results indicate that an antioxidant DPPD can inhibit the early stage of enhanced hepatocarcinogenesis caused by coadministration of ethionine and a CDAA diet, by blocking cellular oxidative damage as well as c-myc and c-Ha-ras expression.
  • K Ohashi; M Tsutsumi; K Kobitsu; T Fukuda; T Tsujiuchi; E Okajima; S Ko; Y Nakajima; H Nakano; Y Konishi
    JAPANESE JOURNAL OF CANCER RESEARCH 87 5 419 - 422 1996年05月 [査読有り]
     
    The telomere length in 20 surgically resected human hepatocellular carcinomas (HCCs) and adjacent non-cancerous livers with hepatitis virus infection were investigated. All the HCC samples examined demonstrated shorter telomere length than the corresponding non-cancerous liver tissues, the respective average values being 5.4 kbp and 8.8 kbp (P < 0.001). The shortening of telomere length was most prominent in HCCs larger than 30 mm in diameter, and in both tumors and non-cancerous livers it was more marked with hepatitis B virus as compared with hepatitis C virus infection. These results indicate that telomere shortening is associated with not only progression, but also development of HCC, and there is a possible difference in the nature of the association in patients with hepatitis viruses of B and C types.
  • H Kitada; M Tsutsumi; T Tsujiuchi; M Takahama; T Fukuda; N Narita; Y Konishi
    MOLECULAR CARCINOGENESIS 15 4 276 - 283 1996年04月 [査読有り]
     
    Point mutations of the Ki-ras and p53 genes in rat lung lesions induced by N-nitrosobis(2-hydroxypropyl)amine (BHP) were investigated by polymerase chain reaction-single-strand conformation polymorphism analysis followed by direct sequencing using paraffin-embedded tissues. Male Wistar rats 6 wk old were given 2000 ppm BHP in drinking water for 15 wk. Another group was given drinking water without BHP. The rats were killed 20-27 wk after the beginning of the experiment. Lung adenomatous and squamous lesions, including carcinomas, were induced. The frequencies of Ki-ras mutations were 40% (six of 15) in alveolar hyperplasias, 36% (five of 14) in adenomas, 72% (18 of 25) in adenocarcinomas, 20% (three of 15) in squamous metaplasias, 50% (three of six) in squamous cell carcinomas, and 50% (five of 10) in adenosquamous carcinomas. The mutations were all G --> A transitions at the second position of codon 12; no other mutations were detected. However, Haras mutations in exons 1 and 2 and p53 mutations in exons 5, 6, and 7 were not detected in adenocarcinomas and squamous cell carcinomas. These results indicate that Ki-ras mutation is an early genetic event in some adenomatous and squamous lung carcinogeneses and that Ki-ras mutations can cause benign lesions to convert to malignant lesions. The results also show that Ha-ras and p53 mutations are not involved in rat lung carcinogenesis induced by BHP. (C) 1996 Wiley-Liss, Inc.
  • T Endoh; Q Tang; A Denda; O Noguchi; E Kobayashi; K Tamura; K Horiguchi; H Ogasawara; T Tsujiuchi; D Nakae; M Sugimura; Y Konishi
    CARCINOGENESIS 17 3 467 - 475 1996年03月 [査読有り]
     
    Effects of inhibitors of arachidonic acid (AA) metabolism on the development of fatty liver, cirrhosis, glutathione-S-transferase placental form (GST-P)-positive nodules and the generation of 8-hydroxydeoxyguanosine (8-OHdG) and thiobarbituric acid-reactive substances (TBARS), caused by a choline-deficient, L-amino acid-defined (CDAA) diet, were examined in male Fischer 344 rats by feeding CDAA diets supplemented with the inhibitors for 12 and 30 weeks, Acetylsalicylic acid (ASA) (at doses of 0.1 and 0.2%) and p-bromophenacylbromide (BPB) (0.1 and 0.2%) were used as inhibitors of, respectively, cyclo-oxygenase and phospholipase A(2), and quercetin (QU) (0.75 and 1.5%) and nordihydroguaiaretic acid (NDGA) (0.1 and 0.2%) as inhibitors of lipoxygenase, None of the inhibitors affected the development of fatty liver caused by the CDAA diet, ASA at a dose of 0.2% almost completely prevented the appearance of cirrhosis, GST-P-positive nodules, 8-OHdG and TBARS in seven out of 11 (63.7%) rats, BPB at a dose of 0.2% also exerted inhibitory effects on all of these lesions but to a lesser extent than ASA, QU and NDGA exerted inhibitory effects limited to the GST-P-positive nodule case, The results indicate that a perturbed AA metabolism, particularly of the cyclo-oxygenase pathway, derived secondarily from depletion of labile methyl groups or phosphatidylcholine, might play key roles in the cirrhosis, hepatocarcinogenesis and oxidative stress caused by a CDAA diet, The results also indicated a possible involvement of the lipoxygenase pathway in hepatocarcinogenic processes.
  • T Tsujiuchi; M Tsutsumi; K Kobitsu; T Majima; A Denda; D Nakae; Y Konishi
    RECENT ADVANCES IN GASTROENTEROLOGICAL CARCINOGENESIS I 513 - 516 1996年 [査読有り]
     
    Activation of telomerase has been reported in several human cancers, including hepatocellular carcinomas (HCCs). We examined telomerase activity during hepatocarcinogenesis induced by a choline deficient L-amino acid defined (CDAA) diet in rats. Male F344 rats, 6-week-old, were given a CDAA diet and sacrificed at 10, 50 and 75 weeks after the beginning of the experiment. Hyperplastic nodules and hepatocellular carcinomas (HCCs) developed in rats fed a CDAA diet for 50 and 75 weeks, respectively. Normal control liver specimens were obtained from 6-weeks-old rats. Telomerase activity was assesed with a telomeric repeat amplification protocol (TRAP) and quantitated with an image analyzer. Normal liver and background parenchyma of the livers at 10, 50 and 75 weeks showed weak telomerase activity. In contrast, relative telomerase activities quantitated to normal liver in hyperplastic nodules and HCCs increased approximately 4.75 and 5.42 times than in normal liver, respectively. These results suggest that increased telomerase activity may be a one of biological phenomena of preneoplastic lesions for the evolution to HCCs in rat liver.
  • A Denda; T Endoh; Q Tang; T Tsujiuchi; D Nakae; Y Konishi
    RECENT ADVANCES IN GASTROENTEROLOGICAL CARCINOGENESIS I 495 - 499 1996年 [査読有り]
     
    A choline-deficient, L-amino acid-defined (CDAA) diet causes hepatocellular carcinomas associated with fatty liver, hepatocyte injury, fibrosis, cirrhosis and oxidative DNA damage in rats. In the present study, possible involvement of a perturbed arachidonic acid (AA) cascade in the causative mechanisms of these lesion-induction by a CDAA diet was examined by feeding CDAA diets containing various inhibitors of AA cascade to 8 week-old F344 male rats for 12 and 30 weeks. The results indicated that relatively strong cyclooxygenase (COX) inhibitors, an irreversible acetylsalicylic acid and long half-life piroxicam but not short half-life indomethacin, can prevent the development of the pathological liver lesions except fatty liver, suggesting that a perturbed AA cascade, particularly augmented COX pathway, might play key roles in the causative mechanisms of the lesion-induction including hepatocarcinogenesis caused by a CDAA diet in rats.
  • Y Konishi; M Tsutsumi; T Tsujiuchi
    RECENT ADVANCES IN GASTROENTEROLOGICAL CARCINOGENESIS I 181 - 185 1996年 [査読有り]
     
    Genetic alteration of pancreatic duct carcinogenesis in hamsters was studied. As molecular events, K-ras but not H-ras and N-ras mutations can be detected as early as ductal hyperplasia and the frequency becomes progressively elevated in atypical hyperplasia to invasive carcinoma. Mutations of p53 were found without allelic loss or MDM2 amplification in transplantable carcinomas and cell lines but not primary carcinomas. Over expressions of nm23, TGF-alpha and VEGF were found in cell lines. Shortened telomere and increased telomerase activity were found in duct carcinoma and cell lines. The results suggest that K-ras mutation is the early event and p53 mutation, increased telomerase activity and the expressions of nm23 and VEGF are late events, of pancreatic duct carcinogenesis in hamsters.
  • K Ohashi; M Tsutsumi; T Tsujiuchi; K Kobitsu; Y Nakajima; H Kanehiro; M Hisanaga; K Nishio; H Nakano; Y Konishi
    RECENT ADVANCES IN GASTROENTEROLOGICAL CARCINOGENESIS I 621 - 624 1996年 [査読有り]
     
    The understanding of genetic alterations in cancers could provide clues to the tumor pathogenesis and prognostic persuading by gene therapy, Ki-ras and p53 gene status in a series of 88 biliary tract carcinomas (BTCs) were investigated in relation to the tumor locations. Mutations at Ki-ras codon 12 were present in 62% and p53 protein overexpressions were present in 33% of the BTCs. The frequency of Ki-ras mutations was highest in BTCs originated from the lower end of the biliary tree and gallbladder carcinomas, while the peripheral intrahepatic cholangiocarcinomas showed the lowest frequency, In contrast, p53 overexpressions did not show remarkable difference in the frequencies according to the tumor locations. These results clearly indicate that the tumor pathogenesis underlined by Ki-ras mutations may differ with the tumor locations. The lower end of ECCs and GBCs should be as good candidates for Ki-ras gene-targeted therapy in future.
  • M Tsutsumi; T Tsujiuchi; K Kobitu; T Majima; Y Konishi
    RECENT ADVANCES IN GASTROENTEROLOGICAL CARCINOGENESIS I 577 - 580 1996年 [査読有り]
     
    Telomere restriction fragment (TRF) length and telomerase activity were investigated in nitrosamine-induced hamster pancreatic ductal adenocarcinomas and cell lines. Analysis of TRF length was performed accoring with procedure of southern blot analysis and telomerase activity was mesured by TRAP assay. As the results, shortning of TRF length and increase of telomerase activity were observed in pancreatic ductal adenocarcinomas and three cell lines. It was suggested that telomere reduction and activation of telomerase might play an imortant role during progression of pancreatic adenocarcinoma The findings may be important basic information for new therapeutic approach to advanced pancreatic duct adenocarcinomas.
  • T Majima; T Tsujiuchi; M Tsutsumi; A Kido; Y Yamamoto; T Tsunoda; Y Konishi
    RECENT ADVANCES IN GASTROENTEROLOGICAL CARCINOGENESIS I 581 - 584 1996年 [査読有り]
     
    The experimental model of pancreaticobiliary maljunction in hamsters, which consists of cholecystoduodenostomy and dissection of the pancreaticobiliary common duct followed by the injections of N-nitrosobis(2-oxopropyl)amine (BOP), could induce not only pancreatic and intrahepatic biliary carcinomas but also extrahepatic biliary and gall bladder carcinomas with high incidence. In the present study, we investigated the mutation of K-ras and p53 genes in pancreatic duct and extrahepatic bile duct carcinomas induced by this model, using with PCR-SSCP analysis and nucleotide direct sequencing analysis. The results showed that 6 out of 8 (75%) extrahepatic biliary carcinomas and 6 of 11 (54.5%) pancreatic duct carcinomas showed the mutation of K-ras exon 1. G to A transition was detected in the second position of codon 12 in both extrahepatic biliary and pancreatic carcinomas. However, no mutations of p53 exon 5 to 7 were detected in both all carcinomas. Therefore, it has been suggested that K-ras gene mutation might play an important role on carcinogenesis in this model.
  • Kazuhiko Yamamoto; Masahiro Tsutsumi; Toshifumi Tsujiuchi; Kunihiko Kobitsu; Eijiro Okajima; Makoto Takahama; Yukio Mori; Kazuhiko Yamamoto; Yukio Mori
    Journal of Toxicologic Pathology 9 2 199 - 204 1996年 [査読有り]
     
    The potential for endogenous formation of N-nitrosobis(2-hydroxypropyl)amine (BHP) and carcinogenicity were investigated in male Wistar rats administered tris(2-hydroxypropyl)amine (THPA) mixed in powdered diet at a concentration of 2% and sodium nitrite dissolved in distilled water at concentrations of 0.15% and 0.3% for 102 weeks. Endogenous formation of BHP was demonstrated by its presence in the urine of rats given 2% THPA and 0.3% nitrite, but not in the groups receiving either of these precursors alone. However, significant induction of tumors was not observed in BHP target organs in groups given 2% THPA together with 0.15% or 0.3% nitrite, nor in groups given precursors alone. In BHP non-target organs, various tumors developed in all groups at levels similar to those previously reported for spontaneous lesions in male Wistar rats. The present results indicate that BHP is endogenously formed in rats given 2% THPA and 0.3% nitrite, but that its amount is not high enough for significant induction of tumors in the target organs of BHP in rats. However, since man is exposed to various precursors for the entire lifespan, we can not exclude the possibility that endogenously formed nitrosamines are a potential risk factor in human cancer development. © 1996, JAPANESE SOCIETY OF TOXICOLOGIC PATHOLOGY. All rights reserved.
  • M Tsutsumi; K Ohashi; T Tsujiuchi; E Kobayashi; K Kobitsu; H Kitada; T Majima; E Okajima; T Endoh; K Hasegawa; T Mori; Y Konishi
    JAPANESE JOURNAL OF CANCER RESEARCH 87 1 5 - 9 1996年01月 [査読有り]
     
    The effect of cell cycle disturbance due to colchicine on the induction of enzyme-altered foci during liver regeneration in rats was studied. For initiation, diethylnitrosamine (DEN) at a dose of 10 mg/kg was injected intraperitoneally and partial hepatectomy (PH) was performed 4 h thereafter. Colchicine at doses of 0, 0.1, 0.25 and 0.5 mg/kg was injected intraperitoneally 1 and 3 days after the initiation, followed by application of selection pressure consisting of 2-acetylaminofluorene (AAF) and carbon tetrachloride (CCl4) administration. As end point lesions, gamma-glutamyltransferase (GGT)-positive enzyme-altered foci were assayed at week 5. There was no significant effect of colchicine on numbers of foci. However, a significant, dose-dependent increase in the area of GGT-positive lesions in the groups treated with colchicine was observed. Bromodeoxyuridine labeling indices were higher in foci induced in colchicine-treated rats than in the untreated rats. In a separate experiment, serum glutamic pyruvic transaminase was not increased significantly after DEN and colchicine treatment, and the mitotic index at 6 days after PH was increased in the liver of colchicine-treated rats. These results suggest that the cell cycle disturbance induced by colchicine causes more pronounced selective growth of cells initiated by DEN and colchicine, and this experimental model may be useful for analyzing the mechanisms underlying that growth advantage and the effects of cell cycle abnormalities in liver carcinogenesis.
  • T Tsujiuchi; E Kobayashi; D Nakae; Y Mizumoto; N Andoh; H Kitada; K Ohashi; T Fukuda; A Kido; M Tsutsumi; A Denda; Y Konishi
    JAPANESE JOURNAL OF CANCER RESEARCH 86 12 1136 - 1142 1995年12月 [査読有り]
     
    The effects of methionine on hepatocarcinogenesis induced by coadministration of a choline-deficient L-amino acid-defined (CDAA) diet and ethionine were examined. F344 male rats were divided into 4 experimental groups. Groups 1 and 2 received the CDAA diet and a choline-supplemented L-amino acid-defined (CSAA) diet, respectively. Group 3 received the CDAA diet containing 0.05% ethionine, and group 4 the CDAA diet containing 0.05% ethionine and 0.47% methionine. Animals were killed after 12 weeks of treatment. Histologically, the CDAA diet induced intracellular fat accumulation and foci. In contrast, ethionine caused not only foci, but also hyperplastic nodules, cholangiofibrosis and the proliferation of oval cells without such fat accumulation. Methionine abolished the development of all of the liver lesions induced by coadministration of the CDAA diet and ethionine. To investigate the effects of methionine on induction of c-myc and c-Ha-ras expression, as well as generation of 8-hydroxyguanine (8-OHGua) and 2-thiobarbituric acid-reacting substances (TEARS), by coadministration of the CDAA diet and ethionine, subgroups of 3 to 5 animals were killed at 2, 4, 8 or 11 days after the beginning of the experiment. Coadministration of the CDAA diet and ethionine markedly enhanced the level of expression of c-myc and c-Ha-ras, 8-OHGua formation and TBARS generation as compared with the CDAA or CSAA diet within 11 days, and methionine blocked these actions. These results indicate that addition of methionine prevents the induction of c-myc and c-Ha-ras expression, 8-OHGua formation and TBARS generation, as well as hepatocellular lesions, by coadministration of the CDAA diet and ethionine in rats, and suggest a possible involvement of oxidative stress and gene expression in hepatocarcinogenesis by these agents.
  • D NAKAE; Y MIZUMOTO; N ANDOH; K TAMURA; K HORIGUCHI; T ENDOH; E KOBAYASHI; T TSUJIUCHI; A DENDA; B LOMBARDI; Y KONISHI
    TOXICOLOGIC PATHOLOGY 23 5 583 - 590 1995年09月 [査読有り]
     
    Groups of female Fischer-344 rats were fed a semipurified choline-deficient (CD) diet, or a semisynthetic L-amino acid-defined choline-deficient (CDAA) diet, for up to 12 wk and effects of the 2 diets on the liver were compared. Steatosis was diffuse and more severe throughout in rats fed the CDAA diet than in rats fed the CD diet. Greater elevations in serum aspartate and alanine aminotransferase activities were also present in the former rats, along with higher 2-bromodeoxyuridine labeling indices in the liver. Discrete amounts of 8-hydroxyguanine were detected in liver DNA, but were not significantly different in rats fed the 2 diets, or from those present in a group of control rats killed at 0 time. Glutathione S-transferase placental form-positive focal lesions were not observed in any of the rats. The results show that the CDAA diet causes more severe degrees of steatosis and liver cell death and proliferation than the CD diet, raising the possibility that it may, in contrast to the CD diet, result in the eventual induction of hepatocellular carcinomas in female Fischer-344 rats.
  • M TSUTSUMI; O NOGUCHI; S OKITA; K HORIGUCHI; E KOBAYASHI; K TAMURA; T TSUJIUCHI; A DENDA; Y KONISHI; K IIMURA; Y MORI
    CARCINOGENESIS 16 3 457 - 459 1995年03月 [査読有り]
     
    The effects of dehydroepiandrosterone sulfate (DHAS), a typical hydroxysteroid sulfotransferase (HSTase) inhibitor, and of 3'-phosphoadenosine 5'-phosphate (PAP), a nonspecific sulfation inhibitor on N-nitrosobis(2-oxopropyl)amine (BOP)-induced initiation were examined in a rapid production model for pancreatic carcinomas in hamsters in order to elucidate the involvement of sulfotransferase in the metabolic activation of beta-oxypropylnitrosamines. While neither low nor high doses of DHAS and PAP exerted any significant influence on the incidence of ductal lesions including carcinomas, the high dose of DHAS (350 mg/kg body wt) and a both low (90 mg/kg) and high (180 mg/kg) doses of PAP reduced the mean numbers of pancreatic ductal adenocarcinomas. The high dose of PAP also reduced the number of all ductal lesions combined, The results thus suggest that metabolic activation with STase is involved in BOP-induced pancreatic ductal carcinogenesis in hamsters, and support the hypothesis that BOP is metabolized to beta-hydroxyalkylnitrosamines followed by activation to proximate sulfuric acid esters by HSTase.
  • K OHASHI; M TSUTSUMI; Y NAKAJIMA; O NOGUCHI; S OKITA; H KITADA; T TSUJIUCHI; E KOBAYASHI; H NAKANO; Y KONISHI
    JAPANESE JOURNAL OF CLINICAL ONCOLOGY 24 6 305 - 310 1994年12月 [査読有り]
     
    The presence of the Ki-ras gene mutations in 14 cases of intrahepatic cholangiocarcinoma (IHC) and nine cases of extrahepatic cholangiocarcinoma (EHC) were investigated by polymerase chain reaction-single strand conformation polymorphism analysis. To obtain enriched tumor cell DNA, the microdissection method was used on formalin-fixed paraffin-embedded tissue sections. Point mutations at codon 12 of the Ki-ras gene were detected in seven (50%) of the 14 cases of IHC and six (67%) of the nine EHC cases. In all but one of the ras gene mutation cases, G to A transitions in the second position of codon 12 were detected, the exception being a G to T transition in the same position in one IHC. No point mutations were detected at codon 13 or 61 in either IHC or EHC. Furthermore, there was no demonstrable correlation between Ki-ras mutation and patient age, tumor Stage, histological findings or prognosis. The present results demonstrated a higher participation of Ki-ras gene mutations in EHC than found in previous studies, and provided a confirmation and extension of the results earlier reported by Tada ef al. and Tsuda et al. for IHC.
  • T TSUJIUCHI; M TSUTSUMI; K HONOKI; K HORIGUCHI; E KOBAYASHI; A DENDA; Y KONISHI
    EXPERIMENTAL AND TOXICOLOGIC PATHOLOGY 46 2 115 - 118 1994年07月 [査読有り]
     
    Effects of novobiocin on the induction of gamma-glutamyltranspeptidase(GGT)-positive foci, an early lesion occurring during hepatocarcinogenesis, after diethylnitrosamine(DEN) initiation were investigated in Fischer 344 rats. Animals were given DEN at a dose of 20 mg/kg b. w. followed by novobiocin at doses of 50, 100 and 200 mg/kg b. w. The latter two doses, but not 50 mg/kg b. w., significantly inhibited the development of GGT-positive foci, providing evidence of the possible involvement of mono(ADP-ribosyl)ation in the initiation phase of hepatocarcinogenesis in rats.
  • H MARUYAMA; T AMANUMA; M TSUTSUMI; T TSUJIUCHI; K HORIGUCHI; A DENDA; Y KONISHI
    CARCINOGENESIS 15 6 1193 - 1196 1994年06月 [査読有り]
     
    The effects of dietary administration of catechol (CC), paramethylcatechol (PMC) and di(2-ethylhexyl)phthalate (DEHP) were compared with that of butylated hydroxyanisole (BHA) in Syrian hamsters initiated with N-nitrosobis(2-hydroxypropyl)amine (BHP). Development of pancreatic atypical hyperplasias and adenocarcinomas in terms of combined multiplicity was significantly reduced by CC and DEHP. A similar slight but non-significant tendency was observed for BHA, while PMC was without effect. No statistically significant reduction of liver or gall bladder lesions was observed. The results thus suggest that both antioxidant and peroxisome proliferator categories of agents can inhibit pancreatic carcinogenesis in hamsters.
  • A DENDA; Q TANG; T ENDOH; T TSUJIUCHI; K HORIGUCHI; O NOGUCHI; Y MIZUMOTO; D NAKAE; Y KONISHI
    CARCINOGENESIS 15 6 1279 - 1283 1994年06月 [査読有り]
     
    Effects of acetylsalicylic acid (ASA) (aspirin) on the pathogenesis of fatty liver, cirrhosis and hepatocarcinogenesis caused by a choline-deficient L-amino acid-defined (CDAA) diet were examined in male Fischer 344 rats fed a CDAA diet supplemented with 0, 0.1, 0.2, 0.4 or 0.8% ASA for 30 weeks. ASA at concentrations of >0.2% prevented the development of both cirrhosis and preneoplastic and neoplastic nodules, but without any directly associated prevention of fatty changes. ASA also prevented hepatocyte proliferation and the generation of thiobarbituric acid-reactive substances and 8-hydroxydeoxyguanosine caused by feeding the CDAA diet, analyzed, respectively, after 1, 12 and 12 weeks. The results clearly indicate that the anti-inflammatory drug ASA, which is not a lipotropic factor, can prevent the pathogenesis of cirrhosis and hepatocarcinogenesis caused by a CDAA diet, which is possibly partly associated with the prevention of reactive oxygen species production.
  • D NAKAE; Y MIZUMOTO; H YOSHIJI; N ANDOH; K HORIGUCHI; K SHIRAIWA; E KOBAYASHI; T ENDOH; N SHIMOJI; K TAMURA; T TSUJIUCHI; A DENDA; Y KONISHI
    JAPANESE JOURNAL OF CANCER RESEARCH 85 5 499 - 505 1994年05月 [査読有り]
     
    The present study was performed to assess the roles of hepatocellular oxidative damage to DNA and constituents other than DNA in rat liver carcinogenesis caused by a choline-deficient, L-amino acid-defined (CDAA) diet by examining the effects of the antioxidant;N,N'-diphenyl-p-phenylenediamine (DPPD). The parameters used for cellular oxidative damage were the level of 8-hydroxyguanine (8-OHGua) for DNA and that of 2 thiobarbituric acid-reacting substance (TBARS) for constituents other than DNA. A total of 40 male Fischer 344 rats, 6 weeks old, were fed the CDAA diet for 12 weeks with or without DPPD (0.05, 0.10 or 0.20%) or butylated hydroxytoluene (BHT, 0.25%). In the livers of the rats, the numbers and sizes of glutathione S-transferase (EC 2.5.1.18) placental form (GSTP)- and/or gamma-glutamyltransferase (GGT, EC 2.3.2.2)-positive lesions and levels of 8-OHGua and TBARS were determined. The GSTP-positive lesions of 0.08 mm(2) or larger were all stained positively for GGT as well in cross-sectional area, whereas the smaller lesions were generally negative for GGT. DPPD and BHT reduced the size of the GSTP-positive lesions without affecting their total numbers. At the same time, they reduced TBARS generation without affecting 8-OHGua formation in DNA. The present results indicate that oxidative DNA damage (represented by 8-OHGua formation) and damage to constituents other than DNA (represented by TBARS generation) may play different roles in rat liver carcinogenesis caused by the CDAA diet; the former appears to be involved in the induction of phenotypically altered hepatocyte populations while the latter may be related to the growth of such populations.
  • M TSUTSUMI; W YASUI; A NAITO; K OHASHI; E KOBAYASHI; O NOGUCHI; K HORIGUCHI; S OKITA; T TSUJIUCHI; H KITADA; O ISHIKAWA; E TAHARA; Y KONISHI
    JAPANESE JOURNAL OF CANCER RESEARCH 85 2 118 - 121 1994年02月 [査読有り]
     
    The expression of cripto gene product was examined immunohistochemically in 45 surgically resected pancreatic tumors, including 32 invasive ductal carcinomas, 4 intraductal papillary adenocarcinomas, 4 intraductal papillary adenomas, 2 mucinous cystadenomas, 2 islet cell tumors, and one solid and cystic tumor, and compared with that in 32 areas of accompanying chronic pancreatitis present in the cases of invasive ductal carcinomas and 5 non-tumorous areas of pancreas without pancreatitis. All pancreatic ductal tumors including adenomas and carcinomas showed positive staining with no difference in terms of staining intensity among intraductal tumors and invasive carcinomas with or without mucin hypersecretion. Islet cell tumors were positively stained but the solid and cystic tumor was negative. Duct epithelial cells and acinar cells were negative but islet cells were positive in the pancreas tissues without pancreatitis. Cells arranged in duct-like structures in areas of accompanying chronic pancreatitis were positively stained. The results suggest that cripto expression might be associated with a growth advantage of tumor cells and also with differentiation to form duct-like structures.
  • Y MIZUMOTO; D NAKAE; H YOSHIJI; N ANDOH; K HORIGUCHI; T ENDOH; E KOBAYASHI; T TSUJIUCHI; N SHIMOJI; A DENDA; T TSUJII; M NAGAO; K WAKABAYASHI; Y KONISHI
    CARCINOGENESIS 15 2 241 - 246 1994年02月 [査読有り]
     
    Effects of a lipophilic derivative of vitamin C, 2-O-octadecylascorbic acid (CV-3611), as well as its parent L-ascorbic acid (AscA), DL-alpha-tocopherol (alpha-T) and its hydrophilic derivative, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), on the number and size of gamma-glutamyltransferase (GGT)-positive putative preneoplastic lesions were examined and compared with their influences on 8-hydroxyguanine formation in DNA and 2-thiobarbituric acid-reacting substance generation in the livers of rats fed a choline-deficient, L-amino acid-defined (CDAA) diet for 12 weeks. A total of 90 male Fischer 344 rats, 6 weeks old, were divided into 18 groups each consisting of five rats. Group 1 received the CDAA diet alone; Groups 2, 3 and 4 received the CDAA diet containing respectively 0.01, 0.05 and 0.10% CV-3611; Groups 5-7, 8-10 and 11-13 similarly received the CDAA diet containing AscA, alpha-T and Trolox, respectively, at these same low, middle and high concentrations; Group 14 received a choline-supplemented, L-amino acid-defined (CSAA) diet alone; Groups 15-18 were given the CSAA diet containing CV-3611, AscA, alpha-T and Trolox, respectively, all at the 0.10% level. While all four vitamin derivatives exerted inhibitory effects on all four parameters, in each case dose-dependently, CV-3611 demonstrated the most pronounced effects. The present results indicated that lipophilic vitamin C derivatives may be particularly effective chemopreventive agents against CDAA diet-associated, oxidative stress-related hepatocarcinogenesis via its superior antioxidative properties.
  • D NAKAE; Y MIZUMOTO; N ANDOH; T ENDOH; E KOBAYASHI; T TSUJIUCHI; A DENDA; Y KONISHI
    FRONTIERS OF REACTIVE OXYGEN SPECIES IN BIOLOGY AND MEDICINE 1058 369 - 370 1994年 [査読有り]
  • A DENDA; T ENDOH; Q TANG; T TSUJIUCHI; O NOGUCHI; N ANDOH; Y MIZUMOTO; D NAKAE; Y KONISHI
    FRONTIERS OF REACTIVE OXYGEN SPECIES IN BIOLOGY AND MEDICINE 1058 491 - 492 1994年 [査読有り]
  • M TSUTSUMI; S KONDOH; O NOGUCHI; K HORIGUCHI; E KOBAYASHI; S OKITA; K OHASHI; K HONOKI; T TSUJIUCHI; Y KONISHI
    JAPANESE JOURNAL OF CANCER RESEARCH 84 11 1101 - 1105 1993年11月 [査読有り]
     
    The presence of K-ras gene mutation was examined in experimentally induced preneoplastic pancreatic ductal lesions. Syrian hamsters received 70 mg/kg of N-nitrosobis(2-oxopropyl)amine (BOP) followed by repeated exposure to an augmentation pressure regimen consisting of choline-deficient diet combined with DL-ethionine and L-methionine and administration of 20 mg/kg BOP. After two augmentation pressure cycles, pancreatic ductal cell hyperplasias appeared and after three cycles, atypical hyperplasias of pancreatic ductal cells and intraductal carcinomas developed. K- ras mutations were detected by single-strand conformation polymorphism analysis of polymerase chain reaction products and nucleotide sequencing. The results showed that K-ras mutation had occurred in one of 9 simple hyperplasias of pancreatic ductal epithelium, in 5 of 9 atypical hyperplasias, and in 4 of 8 intraductal carcinomas. The findings thus suggested that K-ras is activated in association with very early stage malignant transformation of pancreatic ductal cells in hamsters.
  • K HONOKI; M TSUTSUMI; Y MIYAUCHI; Y MII; T TSUJIUCHI; T MORISHITA; S MIURA; M AOKI; E KOBAYASHI; S TAMAI; Y KONISHI
    CANCER RESEARCH 53 20 5038 - 5042 1993年10月 [査読有り]
     
    The relationship between expression of nucleoside diphosphate kinase (NDP kinase)/nm23, c-Ha-ras, and c-myc genes and metastatic potential was assessed in rat-transplantable osteosarcoma lines, derived from spontaneous and chemical carcinogen (4-hydroxyamino quinoline 1-oxide)-induced osteosarcomas in Fischer 344/NS1c rats. These osteosarcomas possess metastatic potential and highly metastatic lines spontaneous osteosarcoma-selected lung metastatic lesions and 4-hydroxyamino-quinoline 1-oxide-induced osteosarcoma-selected lung metastatic lesions were respectively established by selectively transplanting lung metastatic lesions. Northern blot analysis revealed that the levels of NDP kinase/nm23 and c-Ha-ras gene expression were increased in line with metastatic ability; thus transcript levels were remarkably greater in both spontaneous osteosarcoma-selected lung metastatic lesions and 4-hydroxyamino-quinoline 1-oxide-induced osteosarcoma-selected lung metastatic lesions highly metastatic lines than in their respective low metastatic spontaneous and chemical carcinogen (4-hydroxyamino quinoline 1-oxide)-induced osteosarcoma counterparts. c-myc mRNA expression was observed in all tumor lines, without any correlation with metastatic ability. Southern blot analysis did not show evidence of gross rearrangement or amplification of NDP kinase/nm23, c-Ha-ras, or c-myc genes suggesting regulation of their gene expression at the transcriptional and/or posttranscriptional level. These results indicate that NDP kinase/nm23 and c-Ha-ras might be cooperatively involved in a positive manner in signal transduction processes, especially involving G-protein reactions, responsible for metastasis of rat-transplantable osteosarcomas.
  • M TSUTSUMI; Y MURAKAMI; S KONDOH; T TSUJIUCHI; K HOHNOKI; K HORIGUCHI; O NOGUCHI; E KOBAYASHI; S OKITA; T SEKIYA; Y KONISHI
    JAPANESE JOURNAL OF CANCER RESEARCH 84 9 956 - 960 1993年09月 [査読有り]
     
    The presence of K-ras point mutations in pancreatic duct carcinomas and cholangiocarcinomas induced by N-nitrosobis(2-hydroxypropyl)amine (BHP) in Syrian hamsters was investigated by single-strand conformation polymorphism analysis of polymerase chain reaction products from frozen fresh materials in order to clarify the K-ras mutation rates in those two carcinomas induced simultaneously by one carcinogen, BHP. In the examined pancreatic duct carcinomas, 10 out of 16 were positive for a mutation in codon 12 while 3 out of 12 cholangiocarcinomas demonstrated mutation of K-ras gene. G-to-A transition was detected in the second position of codon 12 in both pancreatic carcinomas and cholangiocarcinomas. These results suggest that the role of genetic alteration in carcinogenesis may differ with the target organ, even when initiation is with the same carcinogen.
  • Q TANG; A DENDA; T TSUJIUCHI; M TSUTSUMI; T AMANUMA; Y MURATA; H MARUYAMA; Y KONISHI
    JAPANESE JOURNAL OF CANCER RESEARCH 84 2 120 - 127 1993年02月 [査読有り]
     
    Effects of inhibitors of arachidonic acid (AA) metabolism on the evolution of preneoplastic foci into nodules and of nodules into hepatocellular carcinomas were examined in F344 male rat livers with or without phenobarbital (PB) exposure. p-Bromophenacyl bromide (BPB), acetylsalicylic acid (ASA), and quercetin (QU) were used as inhibitors of phospholipase A2, cyclooxygenase and lipoxygenase, respectively. Preneoplastic liver foci were induced by initiation with N-nitrosodiethylamine (200 mg/kg, i.p.) followed by selection using the procedure of Cayama et al. For the nodule experiment, starting 1 week after completion of the selection procedure, animals bearing foci were given diets containing 0.05% PB plus 0.75, 1, or 1.5% of one of the inhibitors, 0.05% PB alone, or 0.75, 1 or 1.5% of inhibitor alone, or basal diet for 9 weeks. For the carcinoma experiment, 3 weeks after completion of the selection procedure, animals bearing nodules were given the same diets mentioned above for 29 weeks. BPB, ASA and QU either with or without PB accelerated the remodeling of preneoplastic foci, significantly decreasing the numbers of persistent nodules and hyperplastic nodules. ASA either with or without PB significantly decreased the number of hepatocellular carcinomas per rat. BPB and QU, however, significantly decreased the numbers of hepatocellular carcinomas with but not without PB. The results suggested an involvement of AA metabolism in the process of evolution of preneoplastic foci into nodules and hepatocellular carcinomas in rat liver with or without PB exposure.
  • A DENDA; Q TANG; T TSUJIUCHI; M TSUTSUMI; T AMANUMA; Y MURATA; K TAMURA; K HORIGUCHI; D NAKAE; Y KONISHI
    CARCINOGENESIS 14 1 95 - 101 1993年01月 [査読有り]
     
    Effects of oxidative stress induced by redox-enzyme modulation on the progression stage of heptocarcinogenesis were examined by monitoring both hepatocyte injury and hepatocellular carcinoma development in F344 rats bearing preneoplastic liver nodules induced by the Cayama - Farber procedure. Redox-enzyme modulation, which included increased cytochrome P450 reductase activity induced by phenobarbital-Na (100 mg/kg, i.p. for 3 days), inhibition of DT-diaphorase by dicumarol (25 mg/kg, i.p.), depletion of glutathoine by phorone (200 mg/kg, i.p.), supplementation with the Fe(III) sodium salt of EDTA (50 mg/kg, i.p.) and redox-cycling activation by menadione (50 mg/kg, i.g.), exerted no prominent hepatocyte injury within nodules but did cause slight injury in the surrounding hepatocytes in nodule-bearing rats. The same treatments induced severe hepatocyte injury in non-treated normal rats. Redox-enzyme modulation performed every other week for 33 weeks significantly reduced the number of hepatocellular carcinomas developing in nodule-bearing rats. These results indicate that preneoplastic nodules are resistant to the oxidative stress induction caused by redox-enzyme modulation treatment and that, despite toxic effects in surrounding hepatocytes, no progression pressure is exerted. Indeed, the treatment rather demonstrates an inhibitory effect of the evolution of the nodules into hepatocellular carcinomas.
  • K HONOKI; Y DOHI; S TABATA; Y MII; Y MIYAUCHI; M TSUTSUMI; T TSUJIUCHI; T MORISHITA; S MIURA; T MORIYAMA; S TAMAI; Y KONISHI
    MOLECULAR CARCINOGENESIS 7 2 111 - 115 1993年 [査読有り]
     
    Alkaline phosphatase (AP) activity and expression of bone Gla protein (BGP), c-fos, and c-jun were compared in two transplantable osteosarcomas with high potentials for metastasis to the lung. The original spontaneous osteosarcoma (SOS) gradually became histologically undifferentiated, losing its osteogenic activity during serial transfer, whereas the chemical (4-hydroxyaminoquinoline 1-oxide)-induced osteosarcoma (COS) retained osteogenesis. The two osteosarcomas showed similar doubling times and levels of lung metastasis, and strong AP activity was detected on the cell membranes of both. Northern blot analysis revealed that lack of BGP mRNA expression was associated with expression of both c-fos and c-jun proto-oncogenes in SOS. In contrast, neither c-fos nor c-jun mRNAs were detected but BGP mRNA was expressed in the case of COS. These results suggest that the c-fos and c-jun genes may suppress the expression of BGP mRNA relevant to differentiation and osteoid formation in rat osteosarcomas. However, this does not appear to be directly related to proliferative or metastatic biological behavior.
  • T TSUJIUCHI; M TSUTSUMI; K UCHIDA; K HONOKI; S KONDOH; A DENDA; M MIWA; Y KONISHI
    JAPANESE JOURNAL OF CANCER RESEARCH 83 9 985 - 988 1992年09月 [査読有り]
     
    The possibility of poly(ADP-ribosyl)ation playing a role during liver regeneration induced by partial hepatectomy (PH) in vivo was examined. When rats were given an i.p. injection of 3-aminobenzamide (ABA) at a dose of 600 mg/kg body weight 12 h after PH, the levels of DNA synthesis at 20 h after PH were significantly reduced. The time course of DNA synthesis in regenerating liver was significantly delayed in the ABA-treated group. Enzymatic assay revealed the activity of poly(ADP-ribose)polymerase (PADPRP) in controls to be increased in parallel with the increase of DNA synthesis induced by PH. This increase in PADPRP activity was delayed and very much weaker after ABA treatment. The results thus suggested that poly(ADP-ribosyl)ation might play an important role in DNA synthesis during liver regeneration in vivo.
  • H MARUYAMA; T AMANUMA; Y TAKASHIMA; H YOSHIJI; D NAKAE; M TSUTSUMI; T TSUJIUCHI; A DENDA; Y KONISHI
    CARCINOGENESIS 13 8 1365 - 1369 1992年08月 [査読有り]
     
    The modifying effects of an immunosuppressive agent, 6-mercaptopurine (6-MP), on development of focal lesions in liver cirrhosis models induced by carbon tetrachloride (CCl4) or furfural were studied in male F344 rats. Feeding of 6-MP at 50 p.p.m. for 20 weeks to animals with preexisting liver cirrhosis caused immunosuppression, and significantly enhanced the induction of gamma-glutamyl-transpeptidase (GGT)-positive foci and nodules in the CCl4 but not furfural case. Glutathione S-transferase P (GST-P)-positive preneoplastic lesions were not affected. Moreover, phenobarbital (PB) also enhanced the induction of GGT-positive hepatocellular lesions only in the CCl4-induced liver cirrhosis model, no promotion influence being exerted after treatment with the non-carcinogenic furfural. This study, therefore, suggests that 6-MP can enhance the induction of one type of preneoplastic foci and nodules and that essential differences exist between focal lesions arising in cirrhotic livers caused by CCl4 as opposed to furfural.
  • T AMANUMA; H MARUYAMA; T TSUJIUCHI; M TSUTSUMI; A DENDA; Y KONISHI
    CANCER LETTERS 64 2 99 - 107 1992年06月 [査読有り]
     
    Carcinogenic and modification potential of 6-mercaptopurine (6-MP) was studied in a medium-term bioassay system for rat liver carcinogenesis. F344 male rats were initiated with a single dose (200 mg/kg body wt.) of diethylnitrosamine (DEN) i.p. and fed diets containing either 0.005 % or 0.02 % 6-MP with or without 0.05 % phenobarbital (PB) for 6 weeks. Quantitative data revealed that 6-MP did not enhance the appearance of enzyme-altered preneoplastic foci and nodules even when administered at the highest dose (0.02 %) despite showing an immunosuppressive effect and slight liver cell damage. Neither of the doses of 6-MP exerted any significant influence on the enhancing effect of PB when administered simultaneously in the medium-term-bioassay.
  • Yoichi Konishi; Masahiro Tsutsumi; Kazuhiro Mizumoto; Toshihiro Amanuma; Kosuke Horiguchi; Toshifumi Tsujiuchi; Ayumi Denda
    Journal of Toxicologic Pathology 5 2 251 - 255 1992年 [査読有り]
  • K HONOKI; M TSUTSUMI; T TSUJIUCHI; S KONDOH; K SHIRAIWA; Y MIYAUCHI; Y MII; S TAMAI; Y KONISHI; GT BOWDEN
    MOLECULAR CARCINOGENESIS 6 2 122 - 128 1992年 [査読有り]
     
    The expression of the transin, c-fos, and c-jun genes was assessed in transplantable osteosarcomas and malignant fibrous histiocytomas, as well as in pancreatic duct adenocarcinomas and hepatocellular carcinomas of rats and hamsters. Northern blot analysis revealed that both an undifferentiated osteosarcoma of spontaneous origin (SOS) and 4-hydroxyaminoquinoline 1-oxide (4-HAQO)-induced malignant fibrous histiocytomas with metastatic potential to the lung showed remarkably increased expression of transin mRNA transcripts. This was not the case for the other tumors. Interestingly, levels of transin mRNA were lower in lung metastatic lesions than in primary subcutaneous SOS tumors. The primary SOS and MFH expressed both c-fos and c-jun genes in conjunction with the transin gene, whereas the non-transin expressers, a 4-HAQO-induced osteosarcoma (COS) and the pancreatic duct adenocarcinomas, demonstrated one or the other, but not both. These results suggest a possible involvement of transin expression in the progression of spontaneous osteosarcomas and 4-HAQO-induced malignant fibrous histiocytomas in rats. Expression of the c-fos and c-jun genes may play a regulatory role in this process.
  • T TSUJIUCHI; K MIZUMOTO; M TSUTSUMI; A DENDA; T AMANUMA; S KONDOH; Y KONISHI
    CANCER LETTERS 61 1 61 - 66 1991年12月 [査読有り]
     
    Effects of 3-aminobenzamide (ABA) on pancreatic carcinogenesis after initiation by N-nitrosobis(2-oxopropyl)amine (BOP) were investigated in Syrian hamsters. Animals were given BOP at a dose of 70 mg/kg body weight by subcutaneous injection and following a 2-week recovery period, were administered basal diet or basal diet containing 0.5, 0.75 and 1.5 % ABA for 30 weeks. While the incidences of resultant pancreatic lesions, including hyperplasia, atypical hyperplasia and carcinoma, induced by BOP were not significantly influenced by ABA treatment, the mean numbers of those pancreatic lesions were significantly decreased in a dose-dependent way. The results therefore suggested the possible involvement of poly(ADP-ribosyl)ation in the post-initiation phase of pancreatic carcinogenesis in hamsters.
  • T TSUJIUCHI; K MIZUMOTO; M TSUTSUMI; A DENDA; T AMANUMA; S KONDOH; Y KONISHI
    CANCER LETTERS 61 1 61 - 66 1991年12月 
    Effects of 3-aminobenzamide (ABA) on pancreatic carcinogenesis after initiation by N-nitrosobis(2-oxopropyl)amine (BOP) were investigated in Syrian hamsters. Animals were given BOP at a dose of 70 mg/kg body weight by subcutaneous injection and following a 2-week recovery period, were administered basal diet or basal diet containing 0.5, 0.75 and 1.5 % ABA for 30 weeks. While the incidences of resultant pancreatic lesions, including hyperplasia, atypical hyperplasia and carcinoma, induced by BOP were not significantly influenced by ABA treatment, the mean numbers of those pancreatic lesions were significantly decreased in a dose-dependent way. The results therefore suggested the possible involvement of poly(ADP-ribosyl)ation in the post-initiation phase of pancreatic carcinogenesis in hamsters.
  • T TSUJIUCHI; M TSUTSUMI; A DENDA; T AMANUMA; S KONDOH; K KAMINO; Y KONISHI
    JAPANESE JOURNAL OF CANCER RESEARCH 82 7 793 - 799 1991年07月 [査読有り]
     
    The effects of an inhibitor of poly(ADP-ribose)polymerase, 3-aminobenzamide (ABA), on N-nitrosobis(2-hydroxypropyl)amine (BHP)-induced pancreas, liver, gallbladder and lung carcinogenesis in Syrian golden hamsters were investigated. Animals were given either BHP alone, by subcutaneous injection at a dose of 500 mg/kg body weight, or in combination with an intraperitoneal injection of ABA 30 min after the BHP at a dose of 300 or 600 mg/kg body weight once a week for 5 weeks, and then killed 35 weeks after the commencement of the experiment. ABA exerted inhibitory effects on pancreas and lung carcinogenesis induced by BHP, with mean numbers of lesions (including hyperplasias and carcinomas) being significantly decreased compared with the BHP-alone group values, while no significant effect was observed on liver or gallbladder carcinogenesis. These results suggest that the effects of ABA on carcinogenesis depend on the target organ as well as the chemical carcinogen examined.
  • H MARUYAMA; T AMANUMA; D NAKAE; M TSUTSUMI; S KONDO; T TSUJIUCHI; A DENDA; Y KONISHI
    CARCINOGENESIS 12 7 1331 - 1334 1991年07月 [査読有り]
     
    The modifying effects of the phenolic antioxidant catechol (CC) and its analogs hydroquinone (HQ) and resorcinol (RN) on pancreatic carcinogenesis were evaluated in 146 female Syrian golden hamsters. Groups of animals received either saline or 70 mg/kg body wt N-nitrosobis(2-oxopropyl)amine (BOP) s.c. injections, twice with a 2 week interval, followed by basal diet or diet containing 1.5% of CC, HQ or RN, and 0.75% CC from week 4. All hamsters were killed at week 20 and histopathologically examined for development of pancreatic, liver and gall bladder lesions. The total numbers of pancreatic lesions comprising carcinomas, atypical ductal hyperplasias and ductal hyperplasias per hamster were significantly decreased in animals receiving BOP followed by CC, HQ and RN when compared to those in hamsters given BOP followed by basal diet. Incidence values for atypical ductal hyperplasias were also significantly decreased by the RN or 0.75% CC treatments. The results thus suggest that pancreatic carcinogenesis initiated by BOP in Syrian hamsters can be inhibited by treatments with phenolic antioxidants such as CC, HQ and RN for a relatively short experimental period.
  • T TSUJIUCHI; M TSUTSUMI; A DENDA; T AMANUMA; S KONDOH; K KAMINO; Y KONISHI
    JAPANESE JOURNAL OF CANCER RESEARCH 82 7 793 - 799 1991年07月 
    The effects of an inhibitor of poly(ADP-ribose)polymerase, 3-aminobenzamide (ABA), on N-nitrosobis(2-hydroxypropyl)amine (BHP)-induced pancreas, liver, gallbladder and lung carcinogenesis in Syrian golden hamsters were investigated. Animals were given either BHP alone, by subcutaneous injection at a dose of 500 mg/kg body weight, or in combination with an intraperitoneal injection of ABA 30 min after the BHP at a dose of 300 or 600 mg/kg body weight once a week for 5 weeks, and then killed 35 weeks after the commencement of the experiment. ABA exerted inhibitory effects on pancreas and lung carcinogenesis induced by BHP, with mean numbers of lesions (including hyperplasias and carcinomas) being significantly decreased compared with the BHP-alone group values, while no significant effect was observed on liver or gallbladder carcinogenesis. These results suggest that the effects of ABA on carcinogenesis depend on the target organ as well as the chemical carcinogen examined.
  • T AMANUMA; K MIZUMOTO; M TSUTSUMI; T TSUJIUCHI; S KITAZAWA; K HASEGAWA; A DENDA; H MARUYAMA; Y KONISHI
    JAPANESE JOURNAL OF CANCER RESEARCH 82 6 632 - 637 1991年06月 [査読有り]
     
    The potential initiating activity of diethylnitrosamine (DEN) was studied in a rapid production model for pancreatic carcinogenesis in hamsters developed in our laboratory incorporating the principle of selection based on resistance to cytotoxicity, originally demonstrated for liver carcinogenesis in rats. Female Syrian golden hamsters were given DEN at a dose of 100 mg/kg body weight or N-nitrosobis-(2-oxopropyl)amine (BOP) at a dose of 70 mg/kg body weight as initiators followed by 3 cycles of augmentation pressure (choline-deficient diet combined with DL-ethionine, L-methionine upon return to basal diet and then administration of 20 mg/kg body weight BOP), and killed 10 weeks after the beginning of the experiment. DEN followed by the augmentation pressure induced a 65% incidence of total pancreatic lesions including 15% carcinomas, while BOP followed by the augmentation pressure induced 100% incidence of total pancreatic lesions and 84.2% for carcinomas. These yields were significantly greater than those observed for augmentation pressure alone. The results thus indicate that DEN possesses weak initiating activity for pancreatic carcinogenesis under the present experimental conditions.
  • A DENDA; K SAI; Q TANG; T TSUJIUCHI; M TSUTSUMI; T AMANUMA; Y MURATA; D NAKAE; H MARUYAMA; Y KUROKAWA; Y KONISHI
    CARCINOGENESIS 12 4 719 - 726 1991年04月 [査読有り]
     
    Inducibility of oxidative stress in rat liver in vivo by menadione-associated redox cycling activation under redox enzyme modulating conditions was examined by monitoring hepatocyte injury and 8-hydroxydeoxyguanosine (8-OHdG) levels of liver DNA. In addition, the treatment-associated liver tumor initiating activity was assessed in terms of development of gamma-glutamyl-transpeptidase (GGT)- and glutathione S-transferase placental form (GST-P)-positive foci and hyperplastic nodules. With or without following menadione treatment (50 mg/kg, i.g.), redox enzyme modulations of increased cytochrome P450 reductase activity induced by phenobarbital (PB)-Na (100 mg/kg, i.p. for 5 days), inhibition of DT-diaphorase by dicumarol (25 mg/kg, i.p.) and depletion of glutathione by phorone (200 mg/kg, i.p.), with or without further supplement of iron EDTA-Na-Fe(III) (70 mg/kg, i.p.), caused both substantial hepatocyte necrosis and 8-OHdG production in Fisher 344 male rats. Subsequent feeding with a 0.05% PB diet for 64 weeks resulted in slightly increased development of GGT-positive foci but not GST-P positive lesions or hyperplastic nodules, suggesting a lack of tumor-initiating activity of the oxidative DNA damage associated with redox enzyme modulations with or without menadione.
  • T TSUJIUCHI; M TSUTSUMI; A DENDA; S KONDOH; D NAKAE; H MARUYAMA; Y KONISHI
    CARCINOGENESIS 11 10 1783 - 1787 1990年10月 [査読有り]
  • T TSUJIUCHI; M TSUTSUMI; A DENDA; S KONDOH; D NAKAE; H MARUYAMA; Y KONISHI
    CARCINOGENESIS 11 10 1783 - 1787 1990年10月
  • S KITAZAWA; A DENDA; M TSUTSUMI; T TSUJIUCHI; K HASEGAWA; K TAMURA; H MARUYAMA; Y KONISHI
    CARCINOGENESIS 11 8 1323 - 1328 1990年08月 [査読有り]
  • Y KONISHI; K MIZUMOTO; S KITAZAWA; T TSUJIUCHI; M TSUTSUMI; T KAMANO
    INTERNATIONAL JOURNAL OF PANCREATOLOGY 7 1-3 83 - 89 1990年08月 [査読有り]
     
    Two cases of human early pancreatic duct adenocarcinoma were presented, and ductal lesions observed histologically were compared to those induced in hamsters using a rapid-production model of pancreatic carcinoma. In human cases, direct histologic evidence was obtained to suggest that cancerous changes arose from duct epithelial cell hyperplasia, because lesions of hyperplasia and carcinoma coexisted in continuity. In hamster serial-killing studies, it was suggested that carcinoma developed through atypical ductal hyperplasia.
  • M TSUTSUMI; K MIZUMOTO; T TSUJIUCHI; H MARUYAMA; M KOIZUMI; T INAGAKI; M TOYOKAWA; Y KONISHI
    ACTA PATHOLOGICA JAPONICA 40 2 153 - 155 1990年02月 [査読有り]
  • K. Yamamoto; A. Nakajima; H. Eimoto; Y. Takashima; T. Tsujiuchi; M. Sugimura; Y. Konishi
    Experimental Pathology 38 1 53 - 59 1990年 [査読有り]
     
    Dose response to the carcinogenic activity of N-nitrosomethyl(2-hydroxypropyl)amine (MHP) was investigated for the nasal cavity of male Wistar rats. MHP dissolved in 0.9% saline was administered intraperitoneally once a week for 12 weeks at doses of 0,4.4, 8.8 and 17.6 mg/kg body weight, and all surviving animals were sacrificed 26 weeks after the beginning of the experiment. Hyperplasias, papillomas and squamous cell carcinomas were induced. Hyperplasias could be divided into flat, protruding and downgrowth types and were observed in respiratory and squamous epithelial regions. Papillomas and squamous cell carcinomas developed in the respiratory region, and the incidence of the latter reached 83 % in rats which received the highest dose of MHP. These results indicate that MHP shows its carcinogenic activity of the nasal cavity by the development of squamous cell carcinomas. © 1990, VEB Gustav Fischer Verlag Jena. All rights reserved.
  • Masahiro Tsutsumi; Kazuhiro Mizumoto; Toshifumi Tsujiuchi; Hiroshi Maruyama; Masaki Koizumi; Takehiko Inagaki; Motokuni Toyokawa; Yoichi Konishi
    Pathology International 40 2 153 - 155 1990年 [査読有り]
     
    A 77‐year‐old man found to have an esophageal polyp underwent polypectomy. Histopathological examination revealed that the polyp consisted of multiple microcystic structures similar to those observed in cystadenoma of the pancreas. This type of tumor is very rare in the esophagus, and is suggested to originate from esophageal glands or remnants from the embryonal state. Acta Pathol Jpn 40: 153–155. 1990. Copyright © 1990, Wiley Blackwell. All rights reserved
  • K YAMAMOTO; A NAKAJIMA; H EIMOTO; Y TAKASHIMA; T TSUJIUCHI; M SUGIMURA; Y KONISHI
    EXPERIMENTAL PATHOLOGY 38 1 53 - 59 1990年 [査読有り]
  • A DENDA; H URA; T TSUJIUCHI; M TSUTSUMI; H EIMOTO; Y TAKASHIMA; S KITAZAWA; T KINUGASA; Y KONISHI
    CARCINOGENESIS 10 10 1929 - 1935 1989年10月 [査読有り]
  • A DENDA; M TSUTSUMI; T TSUJIUCHI; H EIMOTO; Y KONISHI; S SATO
    CANCER LETTERS 45 1 21 - 26 1989年04月 [査読有り]

書籍

  • ミネルヴァ書房, 『ケアマネジメント用語辞典（改訂版）』
    南 武志; 吉田 繁; 巽 純子; 辻内 俊文; 和田 哲幸; 今西 孝至 (担当:共著範囲:)2007年
  • ミネルヴァ書房, 『ケアマネジメント用語辞典』
    南 武志; 吉田 繁; 巽 純子; 辻内 俊文; 和田 哲幸; 今西 孝至 (担当:共著範囲:)2005年

講演・口頭発表等

  • 膵炎関連痛モデルにおけるメシル酸カモスタットの抗侵害受容的作用．  [通常講演]
    西村 幸容; 川畑 篤史; 関口 富美子; 辻内 俊文; 松波 真帆
    第79回日本薬理学会年会 2010年 横浜 第79回日本薬理学会年会
  • Involvement of endogenous hydrogen sulfide in acute pancreatitis-related pain.  [通常講演]
    西村 幸容; 川畑 篤史; 辻内 俊文; 関口 富美子; 松波 真帆
    6th FENS Forum of European Neuroscience 2008年07月 スイス、ジュネーブ 6th FENS Forum of European Neuroscience
  • Hydrogen sulfide as a nociceptive messenger in rat and mouse pancreata.  [通常講演]
    川畑 篤史; 関口 富美子; 松波 真帆; 辻内 俊文; 西村 幸容
    Neuroscience 2007 2007年11月 アメリカ合衆国、サンディエゴ Neuroscience 2007
     
    4. Kawabata, A., Nishimura, S. 西村幸容, Fukushima, O., Ishikura, H., Takahashi, T., Matsunami, M., Tsujiuchi, T., Sekiguchi, F., Naruse, M., Kamanaka, Y. , 2007, 11, 3-7, Dan Diego, USA.

MISC

共同研究・競争的資金等の研究課題

  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2021年04月 -2024年03月 
    代表者 : 辻内 俊文; 池田 裕子
     
    Gタンパク共役型受容体であるリゾフォスファチジン酸（LPA：lysophosphatidic acid）受容体は、生理活性脂質であるLPAと結合し、細胞増殖・分化・運動・形態形成など様々な細胞応答を呈する。現在までに6種類（LPA1-LPA6）の受容体が同定され、それぞれの受容体は細胞特異的に異なる作用を有することが判明している。がん細胞においても、LPA受容体を介する細胞内シグナルが、がんの発生・増殖・進展に重要な役割を担うことが明らかとなってきている。本研究は、これまでの知見を基盤に、がん細胞の抗がん剤・放射線感受性制御におけるLPA受容体シグナルの分子機構を明らかにするものである。 研究初年度は、種々のがん細胞を用いて抗がん剤処理と放射線照射を行う2つの実験系に大別して研究を行った。 ①抗がん剤処理によるがん細胞の細胞生存率に対するLPA受容体の機能解析 LPA受容体ノックダウン細胞およびLPA受容体agonist・antagonistを用いて、抗がん剤に対するがん細胞の細胞生存率を解析した。シスプラチン（CDDP）処理膵がん細胞では、LPA2を介する細胞内シグナルの活性化が抗がん剤に対する細胞生存率を上昇させた。一方、大腸がん細胞にフルオロウラシル（5-FU）を処理するとLPA4およびLPA6を介する細胞内シグナルが細胞生存率を低下させることがわかった。 ②放射線照射に対するがん細胞のLPA受容体発現レベルの解析 膵がん・肺がん細胞、骨肉腫・線維肉腫細胞に放射線照射（０～15 Gy）を行い、リアルタイムRT-PCR法を用いて遺伝子発現レベルを測定したところ、細胞特異的にLPA受容体遺伝子発現が誘導されることが示された。また、放射線照射に対する細胞生存率は、LPA2を介する細胞内シグナルにより上昇した。
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2018年04月 -2021年03月 
    代表者 : 辻内 俊文; 朴木 寛弥
     
    生理活性脂質のひとつであるリゾフォスファチジン酸（LPA、lysophosphatidic acid）は、Gタンパク質共役型LPA受容体（LPA1-LPA6）に特異的に結合することで様々な細胞応答を誘発する。本研究では、抗がん剤処理によるがん細胞の生物学的特性の変化にどのようにLPA受容体シグナルが関与するかを明らかにする目的で、短期・長期間抗がん剤処理を種々のがん細胞に行い細胞機能への影響を解析した。その結果、抗がん剤処理によるがん細胞の運動・浸潤および抗がん剤に対する細胞生存率の制御にLPA受容体を介する細胞内シグナルが重要な役割を担うことが判明した。
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2015年04月 -2020年03月 
    代表者 : 朴木 寛弥; 藤井 宏真; 辻内 俊文; 藤間 保晶
     
    植物由来ポリフェノールのPterostilbene (PTE)は、骨肉腫細胞株の増殖能を濃度依存性に抑制し、Oct,CD44,NS等の幹細胞マーカーの発現やsphere形成能などその幹細胞特性も抑制し、そのメカニズムがミトコンドリアにおけるOXPHOS抑制によるATP産生量の減少とROS産生の増加によることを見出した。またこの過程にはPDZD8を介したミトコンドリアと粗面小胞体のtetheringが関与していることを見出した。PTEはhonokiol, JQ1, OTX018などのcMyc阻害剤と相乗効果を示し、ミトコンドリア代謝を標的とした癌幹細胞に対する新規薬剤になり得る可能性が示唆する。
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2012年04月 -2015年03月 
    代表者 : 辻内 俊文; 朴木 寛弥; 福嶋 伸之
     
    リゾフォスファチジン酸(LPA)は生理活性物質のひとつで、7回膜貫通型Gタンパク共役受容体であるLPA受容体(LPA1－LPA6)に結合することで様々な細胞応答を制御する。本研究は、がんの浸潤・転移、造腫瘍性、血管新生、抗がん剤耐性などがんの増悪化に6種類のLPA受容体がどのような役割を演じるかを検索した。その結果、LPA受容体はがん細胞の種類によって発現レベルが異なることと、がん細胞特異的にがんの増悪化を促進または抑制することが明らかとなった。本研究成果より、LPA受容体を介するLPAシグナル伝達経路が新規分子標的療法の標的候補分子となりうる可能性が示唆された。
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2012年04月 -2015年03月 
    代表者 : 福嶋 伸之; 辻内 俊文
     
    リゾホスファチジン酸（LPA）は多様な生理活性を示す脂質メディエーターであり、その作用はLPA受容体（LPA1～LPA6）を介して生じる。これまでLPAシグナルががん発生・増殖・進展に関与していることが示唆されており、がん治療の新規標的と考えられている。本研究では、がん組織で発現しているLPA1変異体が異常な細胞内分布を示すこと、その情報伝達も破綻していることを見いだした。また、卵巣がん細胞におけるLPAの作用を検討したところ、LPAシグナルの結果と思われていたがん細胞に対する作用の一部が、その構成成分である脂肪酸に由来するという、新たな知見を得た。
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2009年 -2011年 
    代表者 : 福嶋 伸之; 辻内 俊文; 森山 隆太郎
     
    神経突起の分岐は生後の神経ネットワーク形成に取って必須であるがその機構は未だ不明である。今回我々はリゾホスファチジン酸(LPA)が新規情報伝達経路を介して神経分岐を促進することを示した。神経系細胞にLPA_3受容体を発現させ、LPAで刺激をすると神経分岐の形成が生じた。この作用はG_qシグナル経路の阻害により抑制された。さらに単量体型G蛋白質であるRnd2/Rho7やその効果器であるrapostlinの抑制性変異体を導入してもLPA_3を介した神経分岐形成が阻害された。LPA_3アゴニストの2(S)-OMPTやLPAは初代培養海馬神経細胞の分岐形成を引き起こしたが、この作用もG_qやRnd2の経路の阻害、あるいはLPA_3のノックダウンにより抑制された。これらの知見はLPA_3、G_qおよびRnd2を包括する新規情報伝達経路が神経ネットワーク形成に重要な役割を果たしていることを示唆している。
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2008年 -2010年 
    代表者 : 朴木 寛弥; 辻内 俊文; 田中 康仁; 藤間 保晶
     
    ラットおよびヒト肉腫細胞を用いて、無血清・非足場依存培養により単離された肉腫spheroid細胞('sarcosphere')が、自己複製能・幹細胞関連遺伝子の強発現・細胞の老化に関与する遺伝子発現の低下など幹細胞様性格を持ち、化学療法剤に対し強い抵抗性を有する事を示した。これにはDNA修復酵素の関与が示唆され、幹細胞様細胞の存在が肉腫の薬剤耐性に深く関与する事を示した。肉腫幹細胞を標的とする治療が、肉腫の予後の改善に寄与する可能性が示唆された。
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 1996年 -1999年 
    代表者 : 小西 陽一; 堤 雅弘; 傳田 阿由美; 中江 大; 辻内 俊文
     
    膵癌に関する研究で重要なことは,ヒト膵癌のうちでも最も頻度が高く,且つ予後不良の膵管腺癌を標的とすることである.ヒトの膵管腺癌と組織学的,生物学的更に,遺伝子学的に類似する癌は,実験的にニトロソ化合物を用いてハムスター叉は雑種成犬にのみ発生せしめ得るのみで,他の動物種では発生せしめることは出来ない.本研究は,代表研究者らが開発したハムスターにおける短期膵管腺癌発生系(Mizumoto K. 他,J.Natl. Cancer Inst. 1988 ; 80 : 1564-1567)とこの系により発生した膵管腺癌の培養系(Moti,T. 他,Int. J. Pancreato. 1994 ; 16 : 171-177)を用いて行った. 膵管腺癌の発生と進展に関する遺伝子変化 1.ハムスター膵管腺癌におけるテロメア長の短縮とテロメラーゼ活性増強が検出され,このことはヒト38症例の膵管腺癌組織のうち32例(84%)に活性増強がみられ,このうち20例(53%)はKi-ras突然変異を合併していた. 2.膵管腺癌において増殖因子であるmidkineのnRNAの過剰発現と蛋白レベルでの発現がみられた. 3.膵管腺癌の発生と進展にMMP familiesのうちでMMP-2の発現亢進が重要な因子であることを見出した. 4.膵管腺癌でアポトーシスに関与するbax遺伝子の過剰発現がみられた. 5.膵管腺癌発生に対するb-カテニンの関与は否定された. 制御に関する研究成果 1.8種のヘテロサイクリックアミンのうち,Trp-P-1とDiMeIQXの膵管腺癌発生への関与が示唆された. 2.MMPの阻害剤であるOPB-3206は,膵管発癌を抑制した. 3.0.0025%b-カロチン,0.004%パームカロチンと0.05及び0.5%緑茶ポリフェノール,0.0015%4-hydroxyphenyl retinamide(4-HPR)と0.1%トラネキサムサンは膵管発癌を抑制した. 4.4-HPRとaurapteneは,それぞれ10μMと50μMの濃度で膵管腺癌培養細胞にアポトーシスを誘導した.
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 1995年 -1995年 
    代表者 : 傳田 阿由美; 辻内 俊文
     
    アミノ酸を基質とする抗脂肪肝因子欠乏食(コリン欠乏-低メチオニン-アミノ酸食)(CDAA食)によるラット肝硬変、肝発癌及び酸化性DNA傷害誘発機構へのアラキドン酸(AA)カスケードの関与機構について検索してきた。これまでにcyclooxygenase(COX)阻害剤のaspirinはこれら全ての病変の誘発を抑制し、lipoxygenase阻害剤のnordihydroguaiaretic acid(NDGA)は肝発癌のみを抑制する事、また別のCOX阻害剤のindomethacin(INDM)はいずれも抑制しない事を報告した。本年度は、aspirinとINDMの効果の矛盾を追求する為、他のCOX阻害剤であるpiroxicam(PIROX),ibuprofen(IBUPRO),低用量のINDM,及びaspirinと共に転写因子NF-kB活性化阻害効果が指摘されている抗炎症剤のNa salicylate(SS)とFN-kB活性化阻害剤のpirrolidine dithiocarbamate(PDTC)を用い、これら病変の誘発に対する修飾効果について検索し、以下の結果を得た。 1.PIROX(0.01,0.02,0.04,0.06%)は、0.04%以上の用量にてaspirinと同様にCDAA食によるラット肝硬変、肝前癌性病変、酸化性DNA障害(8-OHdG)及び肝細胞壊死の誘発をいずれも抑制する事が判明した。 2.IBUPRO(0.02,0.04,0.06%)は、0.06%の高用量でのみ肝前癌性病変、8-OHdGを抑制し、肝硬変の線維性隔壁の狭小化を見た。低用量のINDM(0.001,0.002,0.004%)はいずれに対しても修飾効果を示さなかった。 3.SS(0.1,0.2,0.3,0.5%)は用量依存性に肝前癌性病変の誘発を抑制した。 4.PDTC(0.05,0.1,0.2%)は、肝前癌性病変、肝の線維化、8-OHdGの誘発をむしろ亢進せしめた。 5.NDGA(0.2%)は肝細胞壊死の誘発を抑制した。 以上の結果より、COX阻害剤の中で不可逆的阻害剤であるaspirinと半減期が非常に長いPIROXがCDAA食によるラット肝硬変、肝発癌、酸化性DNA傷害、肝細胞壊死の誘発を抑制する事が判明し、CDAA食によるAAカスケードの亢進がこれら病変の誘発に関与し、亢進したAAカスケードを抑制せしめるにはかなり高濃度の強い阻害剤が必要である事が示唆された。また肝発癌機構にはCOX以外にNDGA,SSによっても抑制される別の機構の存在も示唆された。
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 1994年 -1994年 
    代表者 : 傳田 阿由美; 辻内 俊文
     
    抗脂肪肝因子欠乏食であるコリン欠乏アミノ酸(CDAA)食によるラット肝硬変及び肝発癌誘発機構へのアラキドン酸カスケードの関与機構を、種々のアラキドン酸カスケード阻害剤を用い、肝細胞障害の誘発、癌遺伝子発現、活性酸素産生の観点から検索し、以下の結果を得た。 1.Cyclooxygenase(CYO)阻害剤であるaspirinは、CDAA食によるラット肝細胞壊死の誘発を抑制する事が判明し、CDAA食によるラット肝硬変及び肝発癌の誘発に対するaspirinの抑制機構には、肝細胞壊死の抑制が関与している事が示唆された。 2.Aspirinは、CDAA食投与7日の早朝に誘発されるc-myc発現をむしろ亢進せしめる事が判明した。Aspirinによるc-myc発現の亢進と肝細胞壊死の抑制がどのように関与しているのか、今後更に検索する必要がある。 3.CDAA食投与12週におけるc-myc,c-jun,c-Ha-rasの発現に対し、aspirinは有意な抑制効果を示さなかった。CDAA食によるラット肝発癌機構へのc-myc発現の関与が指摘されているが、aspirinによりその発現が抑制される癌遺伝子の検索が今後必要とされる。 4.CYO阻害剤であるindomethacin(INDM)は、aspirinと異なりCDAA食によるラット肝硬変及び肝発癌の誘発に対し有意な抑制効果を示さなかった。Lipoxygenase阻害剤であるnordihydroguaiaretic acid(NDGA)は、quercetinと異なり、CDAA食によるラット肝発癌を軽度から中等度抑制するも、肝硬変は抑制しなかった。活性酸素産生の指標である8-hydroxydeoxyguanosine(8-OHdG)の誘発に対しては、INDMは抑制傾向を示すもNDGAは有意な抑制効果を示さなかった。AspirinとINDMとの作用の相違は重要であり、今後さらに検索する必要がある。
  • 化学発癌機構の解析
  • analysis of mechanism of carcinogenesis by chemical carcinogen

委員歴

  • 1994年   日本病理学会   学術評議員   日本病理学会

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